High throughput assay system

ABSTRACT

The present invention relates to compositions, apparatus and methods useful for concurrently performing multiple, high throughput, biological or chemical assays, using repeated arrays of probes. A combination of the invention comprises a surface, which comprises a plurality of test regions, at least two of which, and in a preferred embodiment, at least twenty of which, are substantially identical, wherein each of the test regions comprises an array of generic anchor molecules. The anchors are associated with bifunctional linker molecules, each containing a portion which is specific for at least one of the anchors and a portion which is a probe specific for a target of interest. The resulting array of probes is used to analyze the presence or test the activity of one or more target molecules which specifically interact with the probes. In a preferred embodiment, a sample to be tested is subjected to a nuclease protection procedure before it is contacted with a combination of the invention.

[0001] This application is a C.I.P. of U.S. application Ser. No.09/337,325, filed on Jun. 21, 1999, which is a C.I.P. of U.S.application Ser. No. 09/218,166, filed on Dec. 22, 1998, which is aC.I.P. of U.S. application Ser. No. 09/109,076, filed on Jul. 2, 1998.This application claims priority of provisional application No.60/068,291, filed Dec. 19, 1997.

FIELD OF THE INVENTION

[0002] This invention relates, e.g., to compositions, apparatus andmethods useful for concurrently performing multiple biological orchemical assays, using repeated arrays of probes. A plurality of regionseach contains an array of generic anchor molecules. The anchors areassociated with bifunctional linker molecules, each containing a portionwhich is specific for at least one of the anchors and a portion which isa probe specific for a target of interest. The resulting array of probesis used to analyze the presence of one or more target molecules whichinteract specifically with the probes. The invention relates to diversefields distinguished by the nature of the molecular interaction,including but not limited to pharmaceutical drug discovery, molecularbiology, biochemistry, pharmacology and medical diagnostic technology.

BACKGROUND OF THE INVENTION

[0003] Pluralities of molecular probes arranged on surfaces or “chips”have been used in a variety of biological and chemical assays. Assaysare performed to determine if target molecules of interest interact withany of the probes. After exposing the probes to target molecules underselected test conditions, detection devices determine whether a targetmolecule has interacted with a given probe.

[0004] These systems are useful in a variety of screening procedures forobtaining information about either the probes or the target molecules.For example, they have been used to screen for peptides or potentialdrugs which bind to a receptor of interest, among others; to screensamples for the presence of, for example, genetic mutations, allelicvariants in a population, or a particular pathogen or strain ofpathogen, among many others; to study gene expression, for example toidentify the mRNAs whose expression is correlated with a particularphysiological condition, developmental stage, or disease state, etc.

DESCRIPTION OF THE INVENTION

[0005] This invention provides compositions, apparatus and methods forconcurrently performing multiple biological or chemical assays, andallows for high throughput analysis of multiple samples—for example,multiple patient samples to be screened in a diagnostic assay, ormultiple potential drugs or therapeutic agents to be tested in a methodof drug discovery. A combination is provided which is useful for thedetection of one or more targets in a sample. This combination comprisesa surface comprising a plurality of spatially discrete regions, whichcan be termed test regions and which can be wells, at least two of whichare substantially identical. Each surface comprises at least two,preferably at least twenty or more, e.g., at least about 25, 50, 96,864, or 1536, etc., of such substantially identical regions. Each testregion defines a space for the introduction of a sample containing (orpotentially containing) one or more targets and contains a biological orchemical array. (Phrases such as “sample containing a target” or“detecting a target in a sample” are not meant to exclude samples ordeterminations (detection attempts) where no target is contained ordetected. In a general sense, this invention involves arrays todetermine whether a target is contained in a sample irrespective ofwhether it is or is not detected.) This array comprises generic“anchors,” each in association with a bifunctional linker molecule whichhas a first portion that is specific for the anchor and a second portionthat comprises a probe which is specific for at least one of thetarget(s). The combination of this invention is placed in contact with asample containing one or more targets, which optionally react with adetector molecule(s), and is then interrogated by a detection devicewhich detects reactions between target molecules and probes in the testregions, thereby generating results of the assay.

[0006] The invention provides methods and compositions particularlyuseful for high throughput biological assays. In especially preferredembodiments, the invention can be used for high throughput screening fordrug discovery. For example, a high throughput assay can be run in many(100 for example) 96-well microplates at one time. Each well of a platecan have, e.g., 36 different tests performed in it by using an array ofabout 36 anchor and linker pairs. That is, 100 plates, with 96 wells perplate, and each with 36 tests per well, can allow for a total of 345,000tests; for example, each of 9,600 different drug candidates can betested simultaneously for 36 different parameters or assays. Highthroughput assays provide much more information for each drug candidatethan do assays which test only one parameter at a time. For example, itis possible in a single initial high throughput screening assay todetermine whether a drug candidate is selective, specific and/ornontoxic. Non-high throughput methods necessitate extensive follow-upassays to test such parameters for each drug candidate of interest.Several types of high throughput screening assays are described, e.g.,in Examples 15-17. The ability to perform simultaneously a wide varietyof biological assays and to do very many assays at once (i.e., in veryhigh throughput) are two important advantages of the invention.

[0007] In one embodiment, for example, using 96-well DNA Bind plates(Coming Costar) made of polystyrene with a derivatized surface for theattachment of primary amines, such as amino acids or modifiedoligonucleotides, a collection of 36 different oligonucleotides can bespotted onto the surface of every well of every plate to serve asanchors. The anchors can be covalently attached to the derivatizedpolystyrene, and the same 36 anchors can be used for all screeningassays. For any particular assay, a given set of linkers can be used toprogram the surface of each well to be specific for as many as 36different targets or assay types of interest, and different test samplescan be applied to each of the 96 wells in each plate. The same set ofanchors can be used multiple times to re-program the surface of thewells for other targets and assays of interest, or it can be re-usedmultiple times with the same set of linkers. This flexibility andreusability represent further advantages of the invention.

[0008] One embodiment of the invention is a combination useful for thedetection of one or more target(s) in a sample, which comprises, beforethe addition of said sample,

[0009] a) a surface, comprising multiple spatially discrete regions, atleast two of which are substantially identical, each region comprising

[0010] b) at least eight different oligonucleotide anchors, each inassociation with

[0011] c) a bifunctional linker which has a first portion that isspecific for the oligonucleotide anchor, and a second portion thatcomprises a probe which is specific for said target(s).

[0012] Another embodiment of the invention is a combination useful forthe detection of one or more target(s) in a sample, which comprises,before the addition of said sample,

[0013] a) a surface, comprising multiple spatially discrete regions, atleast two of which are substantially identical, each region comprising

[0014] b) at least eight different anchors, each in association with

[0015] c) a bifunctional linker which has a first portion that isspecific for the anchor, and a second portion that comprises a probewhich is specific for said target(s).

[0016] Another embodiment of the invention is a method for detecting atleast one target, which comprises contacting a sample which may comprisethe target(s) with a combination as described above, under conditionseffective for said target(s) to bind to said combination. Anotherembodiment is a method for determining an RNA expression pattern, whichcomprises incubating a sample which comprises as target(s) at least twoRNA molecules with a combination as described above, wherein at leastone probe of the combination is a nucleic acid (e.g., oligonucleotide)which is specific (i.e. selective) for at least one of the RNA targets,under conditions which are effective for specific hybridization of theRNA target(s) to the probe(s). Another embodiment is a method foridentifying an agent (or condition(s)) that modulates an RNA expressionpattern, which is the method described above for determining an RNAexpression pattern, further comprising comparing the RNA expressionpattern produced in the presence of said agent (or condition(s)) to theRNA expression pattern produced under a different set of conditions.

[0017] By way of example, FIGS. 1 and 2 illustrate a combination of theinvention and a method of using it to detect an mRNA target. The surfaceof the invention, shown in FIG. 2, contains 15 identical test regions;in an especially preferred embodiment of the invention, each of thesetest regions is a well in a microtiter plate. Each of the test regionscontains six different anchors, here indicated as numbers 1-6. FIG. 1schematically illustrates one of those anchors, anchor 1, which, in amost preferred embodiment of the invention, is an oligonucleotide. Toanchor 1 is attached a linker molecule, linker 1, which comprises twoportions. The first portion, which is specific for the anchor, is inthis illustration an oligonucleotide which can hybridize specifically tothe anchor. The second portion, which is a probe specific for the targetof interest—here, target mRNA 1—is in this illustration anoligonucleotide which can hybridize to that target. Although notillustrated in this figure, each of the remaining five anchors canhybridize to its own linker via the anchor-specific portion; each linkercan contain a probe portion specific for, e.g., an mRNA different from(or the same as) mRNA 1. This illustrated combination can be used toassay as many as 15 different samples at the same time for the presenceof mRNA 1 (or, simultaneously, for mRNA targets which are specified(programmed) by the other five probes in the array). To perform theassay, each sample, which in this example can be an RNA extract from,say, one of 15 independent cell lines, is added in a small volume to oneof the regions, or wells, and incubated under conditions effective forhybridization of the probe and the target. In order to determine if mRNA1 is present in a sample, a detection device which can recognizepatterns, and/or can interrogate specific locations within each regionfor the presence of a signal, is employed. If the cell lines areincubated under conditions in which their mRNAs are labeled in vivo witha tag, and if mRNA 1 is present in a sample, the detector will detect asignal emanating from the tagged mRNA at the location defined byanchor/probe complex 1. Alternatively, the mRNA can be directly labeledin vitro, before or after being added to the regions (wells).Alternatively, as is illustrated in FIG. 1, mRNA can be taggedindirectly, before or after it has hybridized to the probe, e.g., byincubating the RNA with a tagged “detector” oligonucleotide(target-specific reporter oligonucleotide) which is complementary to asequence other than that recognized by the probe. In the illustratedexample, 15 samples can be analyzed simultaneously. Because at least 20or more, e.g., as many as 1536 or more, samples can be analyzedsimultaneously with this invention, it is a very high throughput assaysystem.

[0018] As used herein, “target” refers to a substance whose presence,activity and/or amount is desired to be determined and which has anaffinity for a given probe. Targets can be man-made ornaturally-occurring substances. Also, they can be employed in theirunaltered state or as aggregates with other species. Targets can beattached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. Examples of targets whichcan be employed in this invention include, but are not limited to,receptors (on vesicles, lipids, cell membranes or a variety of otherreceptors); ligands, agonists or antagonists which bind to specificreceptors; polyclonal antibodies, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses, cellsor other materials); drugs; nucleic acids or polynucleotides (includingmRNA, tRNA, rRNA, oligonucleotides, DNA, viral RNA or DNA, ESTs, cDNA,PCR-amplified products derived from RNA or DNA, and mutations, variantsor modifications thereof); proteins (including enzymes, such as thoseresponsible for cleaving neurotransmitters, proteases, kinases and thelike); substrates for enzymes; peptides; cofactors; lectins; sugars;polysaccharides; cells (which can include cell surface antigens);cellular membranes; organelles; etc., as well as other such molecules orother substances which can exist in complexed, covalently bondedcrosslinked, etc. form. As used herein, the terms nucleic acid,polynucleotide, polynucleic acid and oligonucleotide areinterchangeable. Targets can also be referred to as anti-probes.

[0019] As used herein, a “probe” is a substance, e.g., a molecule, thatcan be specifically recognized by a particular target. The types ofpotential probe/target or target/probe binding partners includereceptor/ligand; ligand/antiligand; nucleic acid (polynucleotide)interactions, including DNA/DNA, DNA/RNA, PNA (peptide nucleicacid)/nucleic acid; enzymes, other catalysts, or other substances, withsubstrates, small molecules or effector molecules; etc. Examples ofprobes that are contemplated by this invention include, but are notlimited to, organic and inorganic materials or polymers, includingmetals, chelating agents or other compounds which interact specificallywith metals, plastics, agonists and antagonists for cell membranereceptors, toxins and venoms, viral epitopes, hormones (e.g., opioidpeptides, steroids, etc.), hormone receptors, lipids (includingphospholipids), peptides, enzymes (such as proteases or kinases), enzymesubstrates, cofactors, drugs, lectins, sugars, nucleic acids (includingoligonucleotides, DNA, RNA, PNA or modified or substituted nucleicacids), oligosaccharides, proteins, enzymes, polyclonal and monoclonalantibodies, single chain antibodies, or fragments thereof. Probepolymers can be linear or cyclic. Probes can distinguish betweenphosphorylated and non-phosphorylated proteins, either by virtue ofdifferential activity or differential binding. Probes such as lectinscan distinguish among glycosylated proteins. As used herein, the termsnucleic acid, polynucleotide, polynucleic acid and oligonucleotide areinterchangeable. Any of the substances described above as “probes” canalso serve as “targets,” and vice-versa.

[0020] Any compatible surface can be used in conjunction with thisinvention. The surface (usually a solid) can be any of a variety oforganic or inorganic materials or combinations thereof, including,merely by way of example, plastics such as polypropylene or polystyrene;ceramic; silicon; (fused) silica, quartz or glass, which can have thethickness of, for example, a glass microscope slide or a glass coverslip; paper, such as filter paper; diazotized cellulose; nitrocellulosefilters; nylon membrane; or polyacrylamide or other type of gel pad,e.g., an aeropad or aerobead, made of an aerogel, which is, e.g., ahighly porous solid, including a film, which is prepared by drying of awet gel by any of a variety of routine, conventional methods. Substratesthat are transparent to light are useful when the method of performingan assay involves optical detection. The surface can be of any thicknessor opacity which is compatible with, e.g., conventional methods ofdetection. For example, the surface can be a thick bottom, clearplate,or an opaque plate. In a preferred embodiment, the surface is theplastic surface of a multiwell, e.g., tissue culture dish, for example a24-, 96-, 256-, 384-, 864- or 1536-well plate (e.g., a modified platesuch as a Coming Costar DNA Bind plate). Anchors can be associated,e.g., bound, directly with a surface, or can be associated with one typeof surface, e.g., glass, which in turn is placed in contact with asecond surface, e.g., within a plastic “well” in a microtiter dish. Theshape of the surface is not critical. It can, for example, be a flatsurface such as a square, rectangle, or circle; a curved surface; or athree dimensional surface such as a bead, particle, strand, precipitate,tube, sphere; etc.

[0021] The surface comprises regions which are spatially discrete andaddressable or identifiable. Each region comprises a set of anchors. Howthe regions are separated, their physical characteristics, and theirrelative orientation to one another are not critical. In one embodiment,the regions can be separated from one another by any physical barrierwhich is resistant to the passage of liquids. For example, in apreferred embodiment, the regions can be wells of a multiwell (e.g.,tissue culture) dish, for example a 24-, 96-, 256-, 384-, 864- or1536-well plate. Alternatively, a surface such as a glass surface can beetched out to have, for example, 864 or 1536 discrete, shallow wells.Alternatively, a surface can comprise regions with no separations orwells, for example a flat surface, e.g., piece of plastic, glass orpaper, and individual regions can further be defined by overlaying astructure (e.g,. a piece of plastic or glass) which delineates theseparate regions. Optionally, a surface can already comprise one or morearrays of anchors, or anchors associated with linkers, before theindividual regions are delineated. In another embodiment, arrays ofanchors within each region can be separated from one another by blankspaces on the surface in which there are no anchors, or by chemicalboundaries, such as wax or silicones, to prevent spreading of droplets.

[0022] In yet another embodiment, the regions can be defined as tubes orfluid control channels, e.g., designed for flow-through assays, asdisclosed, for example, in Beattie et al (1995). Clin. Chem. 4, 700-706.Tubes can be of any size, e.g., capillaries or wider bore tubes; canallow the flow of liquids; or can be partially or completely filled witha gel, e.g., agarose or polyacrylamide, through which compounds can betransported (passed through, flowed through, pumped through), e.g., byelectrophoresis; or with a space filling matrix of channels, e.g., oflinear channels, as described, e.g., in Albota et al. (1998). Science281, 1653-1656; Cumpston et al. (1998). Mat. Res. Soc. Symp. Proc. 488,217-225; and/or Cumpston et al. (1999). Nature 398, 51-54. In such aspace-filling matrix, liquid and/or molecules therein can not onlyfollow in direction perpendicular to the wall of the tube, but can alsodiffuse laterally. In a preferred embodiment, a tube is filled with agel or space-filling matrix; the gel or space-filling matrix isactivated for the binding of anchors, and different anchors are passedthrough sequentially, allowing the formation of a an array (e.g., alinear array) of anchors within the gel; and linkers, targets, etc. arepassed through in succession. The array may be linear, 2- or3-dimensional.

[0023] A plurality of assays can be performed in a single tube. Forexample, a single array of anchors, or of anchors in association withlinkers, in a tube can be re-used (e.g., stripped and re-used, orreprogrammed) in sequential assays with the same or different samples.In another embodiment, a plurality of tubes is used in a single assay,e.g., a sample of interest is analyzed in a plurality of tubescontaining different arrays. The anchors and anchor/linker associationsin the tubes can be any of the types described elsewhere herein.

[0024] Regions within or on, etc. a surface can also be defined bymodification of the surface itself. For example, a plastic surface cancomprise portions made of modified or derivatized plastic, which canserve, e.g., as sites for the addition of specific types of polymers(e.g., PEG can be attached to a polystyrene surface and then derivatizedwith carboxyl or amino groups, double bonds, aldehydes, and the like).Alternatively, a plastic surface can comprise molded structures such asprotrusions or bumps, which can serve as platforms for the addition ofanchors. In another embodiment, regions can be gel pads, e.g.,polyacrylamide gel pads or aeropads, which are arrayed in a desiredpattern on a surface such as, e.g., glass, or are sandwiched between twosurfaces, such as, e.g., glass and a quartz plate. Anchors, linkers,etc. can be immobilized on the surface of such pads, or can be imbeddedwithin them. A variety of other arrangements of gel pads on surfaceswill be evident to one of skill in the art, and can be produced byroutine, conventional methods. The relative orientation of the testregions can take any of a variety of forms including, but not limitedto, parallel or perpendicular arrays within a square or rectangular orother surface, radially extending arrays within a circular or othersurface, or linear arrays, etc.

[0025] The spatially discrete regions of the invention are present inmultiple copies. That is, there are at least two, preferably at leasttwenty, or at least about 24, 50, 96, 256, 384, 864, 1536, 2025, ormore, etc., substantially identical, spatially discrete (separated)regions. Increasing numbers of repeated regions can allow for assays ofincreasingly higher throughput. Substantially identical regions, as usedherein, refers to regions which contain identical or substantiallyidentical arrays of anchors and/or anchor/linker complexes.Substantially identical, as used herein, means that an array or regionis intended to serve essentially the same function as another array orregion in the context of analyzing a target in accordance with thisinvention. Differences not essentially affecting function, i.e.,detectability of targets, are along the line of small nucleotideimperfections (omissions/inserts/substitutions) or oligo imperfections(poor surface binding), etc., which do not within assay accuracysignificantly affect target determination results.

[0026] Of course, one of skill in the art will recognize that not all ofthe regions on a surface need to be substantially identical to oneanother. For example, if two different sets of arrays are to be testedin parallel, it might be advantageous to include both sets of arrays ona single surface. For example, the two different sets of arrays can bearranged in alternating striped patterns, to facilitate comparisonbetween them. In another embodiment, the practitioner may wish toinclude regions which can be detected in a distinguishable manner fromthe other regions on the surface and can thereby be used as a“registration region(s).” For example, a registration region cancomprise oligonucleotides or peptides which display a distinctivepattern of fluorescent molecules that can be recognized by a scanningdetection device as a “starting point” for aligning the locations of theregions on a surface.

[0027] The size and physical spacing of the test regions are notlimiting. Typical regions are of an area of about 1 to about 700 mm²,preferably 1 to about 40 mm², and are spaced about 0.5 to about 5 mmapart, and are routinely selected depending on the areas involved. In apreferred embodiment, the regions are spaced approximately 5 mm apart.For example, each region could comprise a rectangular grid, with, forexample, 8 rows and 6 columns, of roughly circular spots of anchorswhich are about 100 micrometers in diameter and 500 micrometers apart;such a region would cover about a 20 millimeter square area. Larger andsmaller region areas and spacings are included.

[0028] The regions can also be further subdivided such that some or allanchors within a region are physically separated from neighboringanchors by means, e.g., of an indentation or dimple. For example, thenumber of subdivisions (subregions) in a region can range from about 10to about 100 or more or less. In one embodiment, a region which is awell of a 1536-well dish can be further subdivided into smaller wells,e.g., about 4 to about 900, preferably about 16 to about 36 wells,thereby forming an array of wells-within-wells. See FIG. 4. Such adimpled surface reduces the tolerance required for physically placing asingle anchor (or group of anchors) into each designated space (locus),and the size of the areas containing anchors is more uniform, therebyfacilitating the detection of targets which bind to the probe.

[0029] The term “anchor” as used herein refers to any entity orsubstance, e.g., molecule, which is associated with (e.g., immobilizedon, or attached either covalently or non-covalently to) the surface, orwhich is a portion of such surface (e.g., derivatized portion of aplastic surface), and which can undergo specific interaction orassociation with a linker or other substance as described herein. Theportion of an anchor which associates with, e.g., a linker molecule, canbe associated with the surface directly, or the anchor can comprise anintermediate “spacer” moiety. Such a spacer can be of any material,e.g., any of a variety of materials which are conventional in the art.In one embodiment, the spacer is a linear carbon molecule having, e.g.,about 5-20 Cs, preferably about 12 Cs. In another embodiment, the spaceris a nucleic acid (of any of the types describes elsewhere herein) whichdoes not undergo specific interaction or association with, e.g., alinker molecule.

[0030] The term “anchor” as used herein can also refer to a group ofsubstantially identical anchors. See, e.g., FIG. 7, which schematicallyrepresents a test region comprising 3 anchors (A, B and C), each ofwhich is present in multiple copies (a “group”). The location of eachgroup of anchors is termed herein a “locus.” As is well known in theart, the number of individual anchor molecules present at a locus islimited only by physical constraints introduced by, e.g., the size ofthe anchors. For example, a locus which is, e.g., about 25-200 μm indiameter, can comprise millions of anchors.

[0031] As used herein, an “anchor/linker complex” exists when an anchorand a linker have combined through molecular association in a specificmanner. The interaction with the linker can be either irreversible, suchas via certain covalent bonds, or reversible, such as via nucleic acidhybridization.

[0032] In a preferred embodiment, the anchor is a nucleic acid, whichcan be of any length (e.g., an oligonucleotide) or type (e.g., DNA, RNA,PNA, or a PCR product of an RNA or DNA molecule). The nucleic acid canbe modified or substituted (e.g., comprising non naturally occurringnucleotides such as, e.g., inosine; joined via various known linkagessuch as sulfamate, sulfamide, phosphorothionate, methylphosphonate,carbamate, etc.; or a semisynthetic molecule such as a DNA-streptavidinconjugate, etc.). Single stranded nucleic acids are preferred.

[0033] A nucleic acid anchor can be of any length which is compatiblewith the invention. For example, the anchor can be an oligonucleotide,ranging from about 8 to about 50 nucleotides in length, preferably about10, 15, 20, 25 or 30 nucleotides. In another embodiment, the anchor canbe as long as about 50 to about 300 nucleotides in length, or longer orshorter, preferably about 200 to about 250 nucleotides. For example, ananchor can comprise about 150 to about 200 nucleotides of “spacer”nucleic acid, as described above, and, adjacent to the spacer, a shortersequence of, e.g., about 10, 15, 20, 25 or 30 nucleotides which isdesigned to interact with a linker molecule (“linker-specificsequence”). Such spacers can be of any length or type of nucleic acid,and can have any base composition which is functional in the invention.In a preferred embodiment, the spacers of each of the anchors at alocus, and/or of the anchors in different loci within a region, aresubstantially identical; the anchors thus differ from one anotherprimarily with regard to their linker-specific sequences.

[0034] Spacers can impart advantages to anchors, allowing for improvedperformance. For example, the linker-specific portions of such an anchorlie further away from the surface, and therefore are less physicallyconstrained and subject to less steric hindrance, than if they werecloser to the surface. This facilitates, for example, the association ofa plurality of different linkers (e.g., about 2 to about 100), havingdifferent target specificities, with the anchors at a given locus. As isdiscussed in more detail below, an individual anchor can comprises (inaddition to a spacer) a plurality of linker-specific sequences which arearranged, e.g., in a tandem linear fashion; this allows for theassociation of a plurality of different types of linkers with at leastone such anchor at a given locus. Also discussed in more detail below isanother way in which a plurality of different types of linkers can beassociated with the anchors at a given locus: at a “mixed locus,” two ormore anchors are each associated with a different linker, having adifferent target specificity. Because of the physical flexibility ofanchors comprising spacers, the anchors at a given locus can readilybind to a plurality of different linker molecules without beingphysically constrained by adjacent anchor molecules. An advantage ofbinding a plurality of linker molecules to the anchors at a given locusis that it allows for the detection of an increased number of targets ata particular locus. In one embodiment, the plurality of linkers bound ata given locus have probes which are specific for different portions ofthe same target nucleic acid of interest (e.g., to differentoligonucleotide sequences within the nucleic acid). This allows foramplified detection of the target compared to detection with a singleprobe. In another embodiment, the plurality of linkers have probes whichare specific for different, e.g. unrelated, targets. This allows for thedetection of a plurality of different targets within a particular locus.A further advantage of anchors comprising spacers is that they can morereadily accommodate linkers which are associated with relatively largemolecules such as, e.g., proteins, and/or which bind to relatively largetargets such as, e.g., proteins, membranes or cells.

[0035] The base composition of a nucleic acid anchor is not necessarilyconstrained. Any base composition of the anchors is acceptable, providedthat the anchors are functional for the purpose of the invention. Forexample, single stranded nucleic acid anchors at a locus, or atdifferent loci in a region, can comprise partially or completely randomsequences (e.g., randomly generated sequences, for example with norestrictions on the relative amounts of A, G, T and/or C). In oneembodiment, the anchors are not “sequence isomers” (e.g., “randomsequence isomers”), i.e., oligonucleotides having identical amounts ofG, C, A and T, but arranged in different relative orders. That is, theanchors in, for example, the different loci of a region do not conformto the equation G_(n) C_(n) A_(m) T_(m), where n and m are integers.See, e.g., the anchors shown in Example 1, which are not random sequenceisomers. In the anchors of the invention, the amounts of G and C do notneed to be approximately the same, nor do the relative amounts of A andT. Furthermore, the net relative amounts of G, C, A and T are notnecessarily constrained. For example, the base composition of theanchors in a region can range from being relatively GC rich (i.e.,greater than 50% G+C), to having equal amounts of G, C, A and T, tobeing relatively AT rich (i.e., greater than 50% A+T). In oneembodiment, the anchors are randomly generated, e.g., in a manner suchthat no constraints are placed on the relative amounts of G, C, A and T.

[0036] Anchors comprising a nucleic acid spacer and one or morelinker-specific portions are unlikely to conform to any particularconstraints on base compostion. For example, if the anchors located atdifferent loci in a region have spacers which are substantiallyidentical, e.g., a substantially identical 25-mer or a 200-mer, but eachanchor has a different linker-specific moiety (e.g., a 25-mer), even ifthe linker-specific moieties meet specific requirements (e.g., thenumber of As and Gs are approximately equal; the number of Ts and Cs areapproximately equal; the oligo conforms to the equation G_(n) C_(n)A_(m) T_(m); and/or that the G+C content meets a particularrequirement), the anchors as a whole will not meet those particularrequirements. Similarly, even if the linker-specific moieties of anchorsat different loci in a region are substantially different from oneanother (e.g., each linker-specific moiety has a sequence which differsby at least about 20%, or 50%, or 80% from each other linker-specificmoiety in the region), the net sequence identities of the anchors,considering the entire length of the nucleic acid, may be far less. Forexample, if each of the anchors comprises a substantially identical250-mer spacer, and a 25-mer linker-specific moiety which is 100%different from every other linker-specific moiety in the region, theanchors will still differ from one another by only 10%.

[0037] An anchor can also be a peptide or a protein. For example, it canbe a polyclonal or monoclonal antibody molecule or fragment thereof, orsingle chain antibody or fragment thereof, which binds specifically tothe portion of a linker that is an antigen or an anti-antibody molecule;in the obverse, the anchor can be a peptide, and the portion of thelinker which binds to it can be an antibody or the like. In anotherembodiment, the anchor can be a lectin (such as concanavalin A oragglutinins from organisms such as Limulus, peanut, mung bean,Phaseolus, wheat germ, etc.) which is specific for a particularcarbohydrate. In another embodiment, the anchor can comprise an organicmolecule, such as a modified or derivatized plastic polymer which canserve, e.g., as the stage for specific solid phase chemical synthesis ofan oligonucleotide. In this case, the derivatized plastic can bedistributed as an array of discrete, derivatized, loci which are formedintegrally into the plastic surface of a combination during themanufacturing process. In another embodiment, the anchor can takeadvantage of specific or preferential binding between metal ions, e.g.,Ni, Zn, Ca, Mg, etc. and particular proteins or chelating agents. Forexample, the anchor can be polyhistidine, and the anchor-specificportion of the linker can be nickel, which is attached via a nickelchelating agent to a target-specific probe. Alternatively, the chelatingagent can be the anchor and the polyhistidine the probe-related portion.Alternatively, the anchor can comprise an inorganic substance. Forexample, it can comprise a metal such as calcium or magnesium, and theanchor-specific portion of the linker can be a preferential chelatingagent, such as EDTA or EGTA, respectively, which is attached to atarget-specific probe. One of skill in the art will recognize that awide range of other types of molecules can also serve as anchors, suchas those general types also discussed in conjunction with probes andtargets.

[0038] An anchor can also be a hybrid structure, such as a DNA duplex,or a duplex comprising, e.g., DNA and protein which interactspecifically in any of the ways described elsewhere herein. For example,the “base moiety” of a duplex anchor (the portion which is in directcontact with the surface) can comprise an optionally modified singlestranded nucleic acid; preferably, the base moiety also comprises aspacer, e.g., a linear carbon spacer as described above. In oneembodiment, a second single stranded nucleic acid is associated with(e.g., hybridized to) this base moiety, to form an anchor whichcomprises at least a partially double stranded (duplex) nucleic acid.For example, the base moiety can comprise a linear carbon spacer whichis attached to the surface at one end, and at the other end is attachedto a single stranded DNA oligonucleotide of about 10-100 nucleotides,preferably about 25 nucleotides; and the second moiety of the duplex cancomprise a sequence which is complementary to at least a portion of thebase moiety, (e.g., to the terminal about 40 nucleotides), followed byan optional spacer (e.g., about 5-15, preferably about 10 nucleotides),followed by a linker-specific sequence (e.g., a sequence of about 8 toabout 50 nucleotides, preferably about 15, 20, 25 or 30 nucleotides,most preferably about 25 nucleotides in length).

[0039] The relative lengths and base compositions of the complementaryportions of an anchor duplex and of its linker-specific sequence(s) canbe varied to suit the needs of an assay, using optimization procedureswhich are conventional in the art. For example, sequences can beselected such that linkers can be dissociated from (e.g., melted apartfrom) duplex anchor molecules under conditions in which the duplexanchors, themselves, remain intact. The remaining arrays of duplexanchors can then be re-used, if desired, to hybridize to the same ordifferent linker molecules. Alternatively, sequences can be selectedsuch that both the anchor/linker hybrids and the two complementaryportions of the duplex anchors are dissociated under the sameconditions, leaving behind only the base moieties in contact with thesurface. In one embodiment, all or substantially all of the basemoieties in a particular locus or in all the loci of a region areidentical, or substantially identical. The arrays of base moietiesremaining after such a dissociation can be re-used (e.g., forhybridization to linker molecules) only if the complementary portions ofthe duplex anchors are first added back, a process which requiresknowledge of the sequence of the base moiety that is involved in duplexformation. The ability to manufacture arrays of anchors which either canor cannot be re-used by a user unfamiliar with the sequence of the basemoieties, represents an advantage of employing such hybrid anchors. Forexample, a manufacturer can prevent unauthorized re-use of its arrays.The prevention of such re-use can also, e.g., forestall problems ofdegraded performance or unreliability occasioned by excessive use.

[0040] In one embodiment, the group of anchors at a given locus within aregion are substantially identical (e.g., are specific for the“anchor-specific” portion of one type of linker, or for one target,only). See, e.g., FIG. 7. In another embodiment, a plurality ofdifferent anchors, having specificities for a plurality of differentlinkers and/or for a plurality of different targets, can be present at agiven locus, called a “mixed locus,” e.g., a plurality of about 2 toabout 100, for example at least about 2, at least about 4 or at leastabout 10. An advantage of mixed loci is that they allow for thedetection of an increased number of different targets at a particularlocus. In one embodiment, each mixed locus contains one anchor which isthe same in every, or at least several, loci. For instance, an anchorwhich is the same in more than one locus can be used for qualityassurance and/or control or for signal normalization.

[0041] Of course, “mixed loci” are also advantageous for surfaces havingonly a single (non-repeated) region. The anchors in each of the loci ofsuch a single region can interact with linkers, or directly with targetsof interest.

[0042] The number of anchors (i.e., groups of anchors at individualloci) in a test region can be at least two, preferably between about 8and about 900 (more or less being included), more preferably betweenabout 8 and about 300, and most preferably between about 30 and about100 (e.g., about 64). In some preferred embodiments, there are about 16,36, 45 or 100 anchors/test region for a surface with 96 test regions(e.g., wells), or about 9, 16 or 25 anchors/test region for a surfacewith 384 test regions (e.g., wells). In a most preferred embodiment,each anchor in a test region has a different specificity from everyother anchor in the array. However, two or more of the anchors can sharethe same specificity and all of the anchors can be identical. In oneembodiment, in which a combination of the invention comprises a verylarge number of test regions (e.g., about 864, 1536, or more), so that alarge number of test samples can be processed at one time, it might ofinterest to test those samples for only a limited number (e.g., about 2,4, 6 or 9) of parameters. In other words, for combinations comprising avery large number of regions, it might be advantageous to have onlyabout 2 to 9 anchors per region.

[0043] The physical spacing and relative orientation of the anchors(i.e., groups of anchors at individual loci) in or on a test region arenot limiting. Typically, the distance between the anchors is about 0.003to about 5 mm or less, preferably between about 0.03 and about 1. Largerand smaller anchor spacings (and areas) are included. The anchors can bearranged in any orientation relative to one another and to theboundaries of the region. For example, they can be arranged in atwo-dimensional orientation, such as a square, rectangular, hexagonal orother array, or a circular array with anchors emanating from the centerin radial lines or concentric rings. The anchors can also be arranged ina one-dimensional, linear array. For example, oligonucleotides can behybridized to specific positions along a DNA or RNA sequence to form asupramolecular array, or in a linear arrangement in a flow through gel,or on the surface of a flow through device or structures within a flowthrough device Alternatively, the anchors can be laid down in a“bar-code”-like formation. (See FIG. 6). For example, anchors can belaid down as long lines parallel to one another. The spacing between orthe width of each long line can be varied in a regular way to yield asimple, recognizable pattern much like a bar-code, e.g., the first andthird lines can be twice as large as the rest, lines can be omitted,etc. An extra empty line can be placed after the last line to demarcateone test region, and the bar code pattern can be repeated in succeedingtest regions.

[0044] The pattern of anchors does not need to be in strict registrywith the positions of the separated assay wells (test regions) orseparate assay droplets. The term “assay positions” will be used torefer to the positions of the assay surface where assay samples areapplied. (These can be defined by the position of separate droplets ofassay sample or by the position of walls or separators definingindividual assay wells on a multi-well plate for example.) The anchorpattern itself (e.g., a “bar code”-like pattern of oligonucleotideanchors) is used to define where exactly each separate anchor ispositioned by pattern recognition—just as each line of a barcode isrecognized by its position relative to the remaining lines. Hence thefirst anchor need not be at one edge or one corner of each assayposition. The first anchor will be found by pattern recognition, ratherthan position relative to the assay position. As long as the area usedby each assay position (the area of the droplet or the area of the wellfor example) is large enough to be certain to contain at least one wholeunit of the repeating pattern of anchors, then each assay point willtest the sample for that assay position for all of the targets specifiedby the (bar-coded) pattern wherever the pattern lies within the area ofthe assay position.

[0045] The anchors do not need to be arranged in a strict or even fixedpattern within each test region. For example, each anchor can beattached to a particle, bead, or the like, which assumes a randomposition within a test region. The location of each anchor can bedetermined by the use, e.g., of a detectable tag. For example, thelinker molecule specific for each type of anchor can be labeled with adifferent fluorescent, luminescent etc. tag, and the position of aparticle comprising a particular linker/anchor pair can be identified bythe nature of the signal emanating from the linker, e.g., the excitationor emission spectrum. One skilled in the art can prepare a set oflinkers with a variety of such attached tags, each with adistinguishable spectrum. Alternatively, the anchors can be labeleddirectly. For example, each type of anchor can be labeled with a tagwhich fluoresces with a different spectrum from the tags on other typesof anchors. Alternatively, the particles, beads or the like can bedifferent from one another in size or shape. Any of the labeling anddetection methods described herein can be employed. For example,fluorescence can be measured by a CCD-based imaging system, by ascanning fluorescence microscope or Fluorescence Activated Cell Sorter(FACS).

[0046] An anchor can interact or become associated specifically with oneportion—the anchor-specific portion—of a linker molecule. By the terms“interact” or “associate”, it is meant herein that two substances orcompounds (e.g., anchor and anchor-specific portion of a linker, a probeand its target, or a target and a target-specific reporter) are bound(e.g., attached, bound, hybridized, joined, annealed, covalently linked,or otherwise associated) to one another sufficiently that the intendedassay can be conducted. By the terms “specific” or “specifically”, it ismeant herein that two components (e.g., anchor and anchor-specificregion of a linker, a probe and its target, or a target and atarget-specific reporter) bind selectively to each other and, in theabsence of any protection technique, not generally to other componentsunintended for binding to the subject components. The parametersrequired to achieve specific interactions can be determined routinely,e.g., using conventional methods in the art.

[0047] For nucleic acids, for example, one of skill in the art candetermine experimentally the features (such as length, base composition,and degree of complementarity) that will enable a nucleic acid (e.g., anoligonucleotide anchor) to hybridize to another nucleic acid (e.g., theanchor-specific portion of a linker) under conditions of selectedstringency, while minimizing non-specific hybridization to othersubstances or molecules (e.g., other oligonucleotide linkers).Typically, the DNA or other nucleic acid sequence of an anchor, aportion of a linker, or a detector oligonucleotide will have sufficientcomplementarity to its binding partner to enable it to hybridize underselected stringent hybridization conditions, and the T_(m) will be about10° to 20° C. above room temperature (e.g., about 37° C.). In general,an oligonucleotide anchor can range from about 8 to about 50 nucleotidesin length, preferably about 15, 20, 25 or 30 nucleotides. As usedherein, “high stringent hybridization conditions” means any conditionsin which hybridization will occur when there is at least 95%, preferablyabout 97 to 100%, nucleotide complementarity (identity) between thenucleic acids. However, depending on the desired purpose, hybridizationconditions can be selected which require less complementarity, e.g.,about 90%, 85%, 75%, 50%, etc. Among the hybridization reactionparameters which can be varied are salt concentration, buffer, pH,temperature, time of incubation, amount and type of denaturant such asformamide, etc. (see, e.g., Sambrook et al. (1989). Molecular Cloning: ALaboratory Manual (2d ed.) Vols. 1-3, Cold Spring Harbor Press, NewYork; Hames et al. (1985). Nucleic Acid Hybridization, IL Press; Daviset al (1986), Basic Methods in Molecular Biology, Elsevir SciencesPublishing, Inc., New York). For example, nucleic acid (e.g., linkeroligonucleotides) can be added to a test region (e.g., a well of amultiwell plate—in a preferred embodiment, a 96 or 384 or greater wellplate), in a volume ranging from about 0.1 to about 100 or more μl (in apreferred embodiment, about 1 to about 50 μl, most preferably about 40μl), at a concentration ranging from about 0.01 to about 5 μM (in apreferred embodiment, about 0.1 μM), in a buffer such as, for example,6×SSPE-T (0.9 M NaCl, 60 mM NaH₂PO₄, 6 mM EDTA and 0.05% Triton X-100),and hybridized to a binding partner (e.g., an oligonucleotide anchor onthe surface) for between about 10 minutes and about at least 3 hours (ina preferred embodiment, at least about 15 minutes) at a temperatureranging from about 4° C. to about 37° C. (in a preferred embodiment, atabout room temperature). Conditions can be chosen to allow highthroughput. In one embodiment of the invention, the reaction conditionscan approximate physiological conditions.

[0048] The design of other types of substances or molecules (e.g.,polypeptides, lectins, etc.) which can, e.g., serve as anchors or asportions of linkers, and the reaction conditions required to achievespecific interactions with their binding partners, are routine andconventional in the art (e.g., as described in Niemeyer et al (1994).Nucl. Acids Res. 22, 5530-5539; Fodor et al (1996). U.S. Pat. No.5,510,270; Pirrung et al (1992), U.S. Pat. No. 5,143,854). Among theincubation parameters are buffer, salt concentration, pH, temperature,time of incubation, presence of carrier and/or agents or conditions toreduce non-specific interactions, etc. For example, to a test region(e.g., a well of a multiwell plate—in a preferred embodiment, a 96 or384 or greater well plate) which contains, as anchors, antibodies, canbe added anti-antibodies (e.g., antigens or antibody-specific secondaryantibodies) in a volume ranging from about 0.1 to about 100 or more μl(in a preferred embodiment, about 1 to about 50 μl, most preferablyabout 40 μl), at a concentration ranging from about 10 pM to about 10 nM(in a preferred embodiment, about 1 nM), in a buffer such as, forexample, 6×SSPE-T, PBS or physiological saline, and incubated with theanchors on the surface for between about 10 minutes and at least about 3hours (in a preferred embodiment, at least about 15 minutes), at atemperature ranging from about 4° C. to about 45° C. (in a preferredembodiment, about 4° C.). For peptide anchors, a length of about 5 toabout 20 amino acids is preferred.

[0049] In some embodiments of the invention, each anchor in an array caninteract with the anchor-specific portion of its corresponding linker tosubstantially the same degree as do the other anchors in the array,under selected reaction conditions. This can insure that the anchorsspecify a substantially uniform array of linkers and, therefore, probes.

[0050] The anchors (i.e., groups of anchors at individual loci) within atest region can be a “generic” set, each anchor of which can interactwith one or more of a variety of different linkers, each having aportion specific to such anchor but with differing “probe” portions;thus, a single array of generic anchors can be used to program or definea varied set of probes. The flexible nature of such a generic assay ofanchors can be illustrated with reference to FIGS. 1 and 2. FIG. 2illustrates a surface which comprises 15 test regions, each of whichcontains an array of 6 different anchors, which in this example can beoligonucleotides. FIG. 1 schematically illustrates one of these(oligonucleotide) anchors, anchor 1, which is in contact with linker 1,which comprises one portion that is specific for anchor 1 and a secondportion that is specific for target mRNA 1. Alternatively, one couldsubstitute, e.g., a linker 2, which, like linker 1, comprises a portionthat is specific for anchor 1, but which comprises a second portion thatis specific for target mRNA 2 instead of target mRNA 1. Thus, anchor 1can be used to specify (or program, or define, or determine) probes foreither of two or more different target mRNAs. The process of generatingand attaching a high resolution pattern (array) of oligonucleotides orpeptides can be expensive, time-consuming and/or physically difficult.The ability to use a pre-formed array of anchors to program a widevariety of probe arrays is one advantage of this invention.

[0051] Although the generic anchors illustrated in FIG. 2 define apattern of oligonucleotide probes, the identical anchor array could alsobe used to program an array of other probes, for example receptorproteins (see, e.g., FIG. 3). Clearly, many permutations are possible,given the range of types of anchor/linker interactions, e.g., even morecomplex layers of “sandwiched” or “piggybacked” probes such asprotein/antibody combinations. For example, in one embodiment a genericset of anchors can be associated (covalently or non-covalently) with aset of linkers to form a modified array of “conjugated” anchors, as isdescribed in more detail below. Thus, the surface of anchors per thisinvention, itself, offers novel advantages.

[0052] In one embodiment of the invention, anchors can interactreversibly with linkers; thus, a generic set of anchors can be re-usedto program a varied set of probes. For example, an oligonucleotideanchor can be separated from the oligonucleotide portion of a linker by,for example, a heating step that causes the two oligonucleotides todissociate, and can then be rebound to a second linker. The ability tore-use anchor arrays, which can be expensive, time-consuming and/orphysically difficult to make, is another advantage of the invention.

[0053] An anchor does not necessarily have to interact with a linker.For example, an anchor can be coupled (directly or indirectly) to adetectable molecule, such as a fluorochrome, and can thereby serve tolocalize a spot within a grid, e.g., for purpose of registration betweenthe test surface and the detector. Alternatively, an anchor can belabeled with a known amount of a detectable molecule so as to serve asinternal quantitation marker, e.g., for purposes of calibration.

[0054] The term “linker” as used herein refers to a bifunctionalsubstance which comprises a first portion (or moiety or part) that isspecific for a chosen (designated) anchor or subset of the anchors(“anchor-specific”) and a second portion that comprises a probe which isspecific for a target of interest (“target-specific”). The two portionsof the linker can be attached via covalent or noncovalent linkages, andcan be attached directly or through an intermediate (e.g., a spacer).

[0055] The chemical nature of the anchor-specific portion of the linkeris, of course, a function of the anchor or anchors with which itinteracts. For example, if the anchor is an oligonucleotide, the portionof the linker which interacts with it can be, for example, a peptidewhich binds specifically to the oligonucleotide, or a nucleic acid whichcan hybridize efficiently and specifically to it under selectedstringent hybridization conditions. The nucleic acid can be, e.g., anoligonucleotide, DNA, RNA, PNA, PCR product, or substituted or modifiednucleic acid (e.g., comprising non naturally-occurring nucleotides suchas, e.g., inosine; joined via various known linkages such as sulfamate,sulfamide, phosphorothionate, methylphosphonate, carbamate; or asemisynthetic molecule such as a DNA-streptavidin conjugate, etc.).Single strand moieties are preferred. The portion of a linker which isspecific for an oligonucleotide anchor can range from about 8 to about50 nucleotides in length, preferably about 15, 20, 25 or 30 nucleotides.If the anchor is an antibody, the portion of the linker which interactswith it can be, e.g., an anti-antibody, an antigen, or a smallerfragment of one of those molecules, which can interact specifically withthe anchor. Substances or molecules which interact specifically with theother types of anchors described above, and which can serve as theanchor-specific portion of a linker, are well-known in the art and canbe designed using conventional procedures (e.g., see above).

[0056] The chemical nature of the target-specific portion of the linkeris, of course, a function of the target for which it is a probe and withwhich it interacts. For example, if the target is a particular mRNA, thetarget-specific portion of the linker can be, e.g., an oligonucleotidewhich binds specifically to the target but not to interfering RNAs orDNAs, under selected hybridization conditions. One of skill in the artcan, using art-recognized methods, determine experimentally the featuresof an oligonucleotide that will hybridize optimally to the target, withminimal hybridization to non-specific, interfering DNA or RNA (e.g., seeabove). In general, the length of an oligonucleotide probe used todistinguish a target mRNA present in a background of a large excess ofuntargeted RNAs can range from about 8 to about 50 nucleotides inlength, preferably about 18, 20, 22 or 25 nucleotides. Anoligonucleotide probe for use in a biochemical assay in which there isnot a large background of competing targets can be shorter. Usingart-recognized procedures (e.g., the computer program BLAST), thesequences of oligonucleotide probes can be selected such that they aremutually unrelated and are dissimilar from potentially interferingsequences in known genetics databases. The selection of hybridizationconditions that will allow specific hybridization of an oligonucleotideprobe to an RNA can be determined routinely, using art-recognizedprocedures (e.g., see above). For example, target RNA [e.g., total RNAor mRNA extracted from tissues or cells grown (and optionally treatedwith an agent of interest) in any vessel, such as the well of amultiwell microtiter plate (e.g., 96 or 384 or more wells)] can be addedto a test region containing a oligonucleotide probe array (see above) ina buffer such as 6×SSPE-T or others, optionally containing an agent toreduce non-specific binding (e.g., about 0.5 mg/ml degraded herring orsalmon sperm DNA, or yeast RNA), and incubated at an empiricallydetermined temperature for a period ranging from between about 10minutes and at least 18 hours (in a preferred embodiment, about 3hours). The stringency of the hybridization can be the same as, or lessthan, the stringency employed to associate the anchors with theanchor-specific portion of the linkers. The design and use of othertypes of probes are also routine in the art, e.g., as discussed above.

[0057] In one embodiment, all, or substantially all, of the linkersassociated with the anchors at a given locus contain an identical (orsubstantially identical) probe, which is specific for a single, specifictarget of interest. In another embodiment, one or more of the linkersassociated with the anchors at a given locus comprises a plurality ofdifferent probes, and thus is specific for a plurality of differenttargets. These probes can be situated in the linker as part of abranched structure, or preferably, can be aligned in a linearrelationship; and they can be of the same material (e.g., are allnucleic acid or are all peptide sequences), or combinations of variousmaterials. In effect, having multiple probes on each linker increasesthe number of targets which can be detected at a particular locus. Inone embodiment, a plurality of probes on given linker are all specificfor a particular target of interest (e.g., they are specific fordifferent portions of a single mRNA of interest, or are specific fornuclease protection fragments corresponding to different portions ofthat mRNA); this allows for increased sensitivity of an assay for thetarget, e.g., a target which is present in a sample at low abundance.The number of probes on a linker can be, e.g., about 2-50, preferablyabout 2, 4 or 10.

[0058] Of course, linkers which comprise such a plurality of differentprobes are also advantageous for use with surfaces that contain only asingle (non-repeated) region.

[0059] The anchor-specific and the target-specific portions of a linkercan be joined (attached, linked) by any of a variety of covalent ornon-covalent linkages, the nature of which is not essential to theinvention. The two portions can be joined directly or through anintermediate molecule. In one embodiment, in which both portions of thelinker are oligonucleotides, they can be joined by covalent linkagessuch as phosphodiester bonds to form a single, colinear nucleic acid. Inanother embodiment, in which the anchor-specific portion is anoligonucleotide and the target-specific portion is a receptor, forexample a receptor protein, the two portions can be joined via theinteraction of biotin and streptavidin molecules, an example of which isillustrated in FIG. 3. Many variations of such linkages are known (e.g.,see Niemeyer et al (1994). NAR 22, 5530-5539). Alternatively, the twoportions can be joined directly, e.g., an oligonucleotide can beamidated and then linked directly (e.g., crosslinked) to a peptide orprotein via an amide bond, or joined to a membrane component via anamide bond or a lipid attachment. Methods to form such covalent ornoncovalent bonds are conventional and are readily optimized by one ofskill in the art. Spacer sequences (e.g., nucleic acid) can also bepresent between the anchor-specific and target-specific portions of alinker.

[0060] After two substances are associated (e.g., by incubation of twonucleic acids, two proteins, a protein plus a nucleic acid, or others)to form a complex (such as, e.g., an anchor/linker complex), theresulting complex can be optionally treated (e.g., washed) to removeunbound substances (e.g., linkers), using conditions which aredetermined empirically to leave specific interactions intact, but toremove non-specifically bound material. For example, reaction mixturescan be washed between about one and ten times or more under the same orsomewhat more stringent conditions than those used to achieve thecomplex (e.g., anchor/linker complex).

[0061] One of skill in the art will recognize that a variety of types ofsandwiches of anchors and linkers can be generated. For example, to anarray of anchors (e.g., anchors having substantially identicalsequences), one can attach a first set of linkers, each of which has afirst moiety that is specific for the anchor and a second moiety that isspecific for one of a second set of linkers, and so forth. In effect,this second layer of a sandwich allows one to convert a first set ofanchors (e.g., identical oligonucleotides) to a different array having adifferent set of specificities, of “conjugated” anchors. The varioussets of linkers and anchors can be associated to one another covalentlyor non-covalently, as desired.

[0062] The combinations of this invention can be manufactured routinely,using conventional technology.

[0063] Some of the surfaces which can be used in the invention arereadily available from commercial suppliers. In a preferred embodiment,the surface is a 96-, 384- or 1536-well microtiter plate such asmodified plates sold by Corning Costar. Alternatively, a surfacecomprising wells which, in turn, comprise indentations or “dimples” canbe formed by micromachining a substance such as aluminum or steel toprepare a mold, then microinjecting plastic or a similar material intothe mold to form a structure such as that illustrated in FIG. 4.Alternatively, a structure such as that shown in FIG. 4, comprised ofglass, plastic, ceramic, or the like, can be assembled, e.g., from threepieces such as those illustrated in FIG. 5: a first section, called awell separator (FIG. 5a), which will form the separations between thesample wells; a second section, called a subdivider (FIG. 5b), whichwill form the subdivisions, or dimples, within each test well; and athird section, called a base (FIG. 5c), which will form the base of theplate and the lower surface of the test wells. The separator can be, forexample, a piece of material, e.g., silicone, with holes spacedthroughout, so that each hole will form the walls of a test well whenthe three pieces are joined. The subdivider can be, for example, a thinpiece of material, e.g., silicone, shaped in the form of a screen orfine meshwork. The base can be a flat piece of material, e.g., glass,in, for example, the shape of the lower portion of a typical microplateused for a biochemical assay. The top surface of the base can be flat,as illustrated in FIG. 5c, or can be formed with indentations that willalign with the subdivider shape to provide full subdivisions, or wells,within each sample well. The three pieces can be joined by standardprocedures, for example the procedures used in the assembly of siliconwafers.

[0064] Oligonucleotide anchors, linker moieties, or detectors can besynthesized by conventional technology, e.g., with a commercialoligonucleotide synthesizer and/or by ligating together subfragmentsthat have been so synthesized. Nucleic acids which are too long to becomfortably synthesized by such methods can be generated byamplification procedures, e.g., PCR, using conventional procedures. Inone embodiment of the invention, preformed nucleic acid anchors, such asoligonucleotide anchors, can be situated on or within the surface of atest region by any of a variety of conventional techniques, includingphotolithographic or silkscreen chemical attachment, disposition by inkjet technology, capillary, screen or fluid channel chip, electrochemicalpatterning using electrode arrays, contacting with a pin or quill, ordenaturation followed by baking or UV-irradiating onto filters (see,e.g., Rava et al (1996). U.S. Pat. No. 5,545,531; Fodor et al (1996).U.S. Pat. No. 5,510,270; Zanzucchi et al (1997). U.S. Pat. No.5,643,738; Brennan (1995). U.S. Pat. No. 5,474,796; PCT WO 92/10092; PCTWO 90/15070). Anchors can be placed on top of the surface of a testregion or can be, for example in the case of a polyacrylamide gel pad,imbedded within the surface in such a manner that some of the anchorprotrudes from the surface and is available for interactions with thelinker. In a preferred embodiment, preformed oligonucleotide anchors arederivatized at the 5′ end with a free amino group; dissolved at aconcentration routinely determined empirically (e.g., about 1 μM) in abuffer such as 50 mM phosphate buffer, pH 8.5 and 1 mM EDTA; anddistributed with a Pixus nanojet dispenser (Cartesian Technologies) indroplets of about 10.4 nanoliters onto specific locations within a testwell whose upper surface is that of a fresh, dry DNA Bind plate (ComingCostar). Depending on the relative rate of oligonucleotide attachmentand evaporation, it may be required to control the humidity in the wellsduring preparation. In another embodiment, oligonucleotide anchors canbe synthesized directly on the surface of a test region, usingconventional methods such as, e.g., light-activated deprotection ofgrowing oligonucleotide chains (e.g., in conjunction with the use of asite directing “mask”) or by patterned dispensing of nanoliter dropletsof deactivating compound using a nanojet dispenser. Deprotection of allgrowing sequences that are to receive a single nucleotide can be done,for example, and the nucleotide then added across the surface. Inanother embodiment, oligonucleotide anchors are attached to the surfacevia the 3′ ends of the oligonucleotides, using conventional methodology.

[0065] Peptides, proteins, lectins, chelation embodiments, plastics andother types of anchors or linker moieties can also be routinelygenerated, and anchors can be situated on or within surfaces, usingappropriate available technology (see, e.g., Fodor et al (1996). U.S.Pat. No. 5,510,270; Pirrung et al (1992). U.S. Pat. No. 5,143,854;Zanzucchi et al (1997). U.S. Pat. No. 5,643,738; Lowe et al (1985). U.S.Pat. No. 4,562,157; Niemeyer et al (1994). NAR 22, 5530-5539).

[0066] In some embodiments of the invention, the disclosed combinationsare used in a variety of screening procedures and/or to obtaininformation about the level, activity or structure of the probes ortarget molecules. Such assays are termed Multi Array Plate Screen (MAPS)methods or assays, and the surfaces comprising arrays of anchors oranchors plus probes which are used for the assays are termed MAPS arraysor MAPS plates.

[0067] The components of a reaction mixture, assay, or screeningprocedure can be assembled in any order. For example, the anchors,linkers and targets can be assembled sequentially; or targets andlinkers, in the presence or absence of reporters, can be assembled insolution and then contacted with the anchors.

[0068] One embodiment of the invention relates to a method of detectingat least one target, comprising

[0069] a) contacting a sample which may comprise said target(s) with abifunctional linker which has a first portion that is specific for anoligonucleotide anchor and a second portion that comprises a probe whichis specific for said target(s), under conditions effective to obtain afirst hybridization product between said target(s) and said linker,

[0070] b) contacting said first hybridization product with a combinationunder conditions effective to obtain a second hybridization productbetween said first hybridization product and said combination, whereinsaid combination comprises, before the addition of said firsthybridization product,

[0071] 1) a surface comprising multiple spatially discrete regions, atleast two of which are substantially identical, each region comprising

[0072] 2) at least 8 different oligonucleotide anchors,

[0073] c) contacting said first hybridization product or said secondhybridization product with a labeled detector probe, and

[0074] d) detecting said detection probe.

[0075] Each of the assays or procedures described below can be performedin a high throughput manner, in which a large number of samples (e.g.,as many as about 864, 1036, 1536, 2025 or more, depending on the numberof regions in the combination) are assayed on each plate or surfacerapidly and concurrently. Further, many plates or surfaces can beprocessed at one time. For example, in methods of drug discovery, alarge number of samples, each comprising a drug candidate (e.g., amember of a combinatorial chemistry library, such as variants of smallmolecules, peptides, oligonucleotides, or other substances), can beadded to separate regions of a combination as described or can be addedto biological or biochemical samples that are then added to separateregions of a combination, and incubated with probe arrays located in theregions; and assays can be performed on each of the samples. With therecent advent and continuing development of high-density microplates,DNA spotting tools and of methods such as laser technology to generateand collect data from even denser microplates, robotics, improveddispensers, sophisticated detection systems and data-managementsoftware, the methods of this invention can be used to screen or analyzethousands or tens of thousands or more of compounds per day.

[0076] For example, in embodiments in which the probes areoligonucleotides, the assay can be a diagnostic nucleic acid orpolynucleotide screen (e.g., a binding or other assay) of a large numberof samples for the presence of genetic variations or defects (e.g.,polymorphisms or specific mutations associated with diseases such ascystic fibrosis. See, e.g., Iitia et al (1992). Molecular and CellularProbes 6, 505-512)); pathogenic organisms (such as bacteria, viruses,and protozoa, whose hosts are animals, including humans, or plants), ormRNA transcription patterns which are diagnostic of particularphysiological states or diseases. Nucleic acid probe arrays comprisingportions of ESTs (including full-length copies) can be used to evaluatetranscription patterns produced by cells from which the ESTs werederived (or others). Nucleic acid probes can also detect peptides,proteins, or protein domains which bind specifically to particularnucleic acid sequences (and vice-versa).

[0077] Similarly, in embodiments in which the probes are antigen-bindingmolecules (e.g., antibodies), the assay can be a screen for variantproteins, or for protein expression patterns which are diagnostic forparticular physiological states or disease conditions. See, e.g., FIGS.40 and 41 for illustrations of the types of molecules which can bedetected.

[0078] In another embodiment, the combinations of the invention can beused to monitor biochemical reactions such as, e.g., interactions ofproteins, nucleic acids, small molecules, or the like—for example theefficiency or specificity of interactions between antigens andantibodies; or of receptors (such as purified receptors or receptorsbound to cell membranes) and their ligands, agonists or antagonists; orof enzymes (such as proteases or kinases) and their substrates, orincreases or decreases in the amount of substrate converted to aproduct; as well as many others. Such biochemical assays can be used tocharacterize properties of the probe or target, or as the basis of ascreening assay. For example, to screen samples for the presence ofparticular proteases (e.g., proteases involved in blood clotting such asproteases Xa and VIIa), the samples can be assayed on combinations inwhich the probes are fluorogenic substrates specific for each proteaseof interest. If a target protease binds to and cleaves a substrate, thesubstrate will fluoresce, usually as a result, e.g., of cleavage andseparation between two energy transfer pairs, and the signal can bedetected. In another example, to screen samples for the presence of aparticular kinase(s) (e.g., Src, tyrosine kinase, or ZAP70), samplescontaining one or more kinases of interest can be assayed oncombinations in which the probes are peptides which can be selectivelyphosphorylated by one of the kinases of interest. Using art-recognized,routinely determinable conditions, samples can be incubated with thearray of substrates, in an appropriate buffer and with the necessarycofactors, for an empirically determined period of time. (In someassays, e.g., for biochemical studies of factors that regulate theactivity of kinases of interest, the concentration of each kinase can beadjusted so that each substrate is phosphorylated at a similar rate.)After treating (e.g., washing) each reaction under empiricallydetermined conditions to remove kinases and undesired reactioncomponents (optionally), the phosphorylated substrates can be detectedby, for example, incubating them with detectable reagents such as, e.g.,fluorescein-labeled anti-phosphotyrosine or anti-phosphoserineantibodies (e.g., at a concentration of about 10 nM, or more or less),and the signal can be detected. In another example, binding assays canbe performed. For example, SH2 domains such as GRB2 SH2 or ZAP70 SH2 canbe assayed on probe arrays of appropriate phosphorylated peptides; orblood sera can be screened on probe arrays of particular receptors forthe presence of immune deficiencies. Also, enzyme-linked assays can beperformed in such an array format. Combinations of the invention canalso be used to detect mutant enzymes, which are either more or lessactive than their wild type counterparts, or to screen for a variety ofagents including herbicides or pesticides.

[0079] Of course, MAPS assays can be used to quantitate (measure,quantify) the amount of active target in a sample, provided that probeis not fully occupied, that is, not more than about 90% of availableprobe sites are bound (or reacted or hybridized) with target. Underthese conditions, target can be quantitated because having more targetwill result in having more probe bound. On the other hand, underconditions where more than about 90% of available probe sites are bound,having more target present would not substantially increase the amountof target bound to probe. Any of the heretofore-mentioned types oftargets can be quantitated in this manner. For example, Example 6describes the quantitation of oligonucleotide targets. Furthermore, itdemonstrates that even if a target is present in large excess (e.g., ifit is present in such large amounts that it saturates the amount ofavailable probe in a MAPS probe array), by adding known amounts ofunlabeled target to the binding mixture, one can “shift the sensitivity”of the reaction in order to allow even such large amounts of target tobe quantitated.

[0080] In another embodiment, combinations of the invention can be usedto screen for agents which modulate the interaction of a target and agiven probe. An agent can modulate the target/probe interaction byinteracting directly or indirectly with either the probe, the target, ora complex formed by the target plus the probe. The modulation can take avariety of forms, including, but not limited to, an increase or decreasein the binding affinity of the target for the probe, an increase ordecrease in the rate at which the target and the probe bind, acompetitive or non-competitive inhibition of the binding of the probe tothe target, or an increase or decrease in the activity of the probe orthe target which can, in some cases, lead to an increase or decrease inthe probe/target interaction. Such agents can be man-made ornaturally-occurring substances. Also, such agents can be employed intheir unaltered state or as aggregates with other species; and they canbe attached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. For example, to identifypotential “blood thinners,” or agents which interact with one of thecascade of proteases which cause blood clotting, cocktails of theproteases of interest can be tested with a plurality of candidate agentsand then tested for activity as described above. Other examples ofagents which can be employed by this invention are very diverse, andinclude pesticides and herbicides. Examples 16 and 17 describe highthroughput assays for agents which selectively inhibit specific kinases,or for selective inhibitors of the interaction between SH2 domains andphosphorylated peptides.

[0081] In another embodiment, the combinations of the invention can beused to screen for agents which modulate a pattern of gene expression.Arrays of oligonucleotides can be used, for example, to identify mRNAspecies whose pattern of expression from a set of genes is correlatedwith a particular physiological state or developmental stage, or with adisease condition (“correlative” genes, RNAs, or expression patterns).By the terms “correlate” or “correlative,” it is meant that thesynthesis pattern of RNA is associated with the physiological conditionof a cell, but not necessarily that the expression of a given RNA isresponsible for or is causative of a particular physiological state. Forexample, a small subset of mRNAs can be identified which are expressed,upconverted and/or downconverted in cells which serve as a model for aparticular disease state; this altered pattern of expression as comparedto that in a normal cell, which does not exhibit a pathologicalphenotype, can serve as a indicator of the disease state (“indicator”genes, RNAs, or expression patterns). The terms “correlative” and“indicator” can be used interchangeably. For example, cells treated witha tumor promoter such as phorbol myristate might exhibit a pattern ofgene expression which mimics that seen in the early stages of tumorgrowth. In another model for cancer, mouse insulinoma cells (e.g., cellline TGP61), when infected with adenovirus, exhibit an increase in theexpression of, e.g., c-Jun and MIP-2, while the expression ofhousekeeping genes such as GAPDH and L32 remains substantiallyunaffected.

[0082] Agents which, after contacting a cell from a disease model,either directly or indirectly, and either in vivo or in vitro (e.g., intissue culture), modulate the indicator expression pattern, might act astherapeutic agents or drugs for organisms (e.g., human or other animalpatients, or plants) suffering from the disease. Agents can alsomodulate expression patterns by contacting the nucleic acid directly,e.g., in an in vitro (test tube) expression system. As used herein,“modulate” means to cause to increase or decrease the amount and/oractivity of a molecule or the like which is involved in a measurablereaction. The combinations of the invention can be used to screen forsuch agents. For example, a series of cells (e.g., from a disease model)can be contacted with a series of agents (e.g., for a period of timeranging from about 10 minutes to about 48 hours or more) and, usingroutine, art-recognized methods (e.g., commercially available kits),total RNA or mRNA extracts can be made. If it is desired to amplify theamount of RNA, standard procedures such as RT-PCR amplification can beused (see, e.g., Innis et al eds., (1996) PCR Protocols: A Guide toMethods in Amplification, Academic Press, New York). The extracts (oramplified products from them) can be allowed to contact (e.g., incubatewith) a plurality of substantially identical arrays which compriseprobes for appropriate indicator RNAs, and those agents which areassociated with a change in the indicator expression pattern can beidentified. Example 15 describes a high throughput assay to screen forcompounds which may alter the expression of genes that are correlativewith a disease state.

[0083] Similarly, agents can be identified which modulate expressionpatterns associated with particular physiological states ordevelopmental stages. Such agents can be man-made or naturally-occurringsubstances, including environmental factors such as substances involvedin embryonic development or in regulating physiological reactions, orsubstances important in agribusiness such as pesticides or herbicides.Also, such agents can be employed in their unaltered state or asaggregates with other species; and they can be attached, covalently ornoncovalently, to a binding member, either directly or via a specificbinding substance.

[0084] Another embodiment of the invention is a kit useful for thedetection of at least one target in a sample, which comprises:

[0085] a) a surface, comprising multiple spatially discrete regions, atleast two of which are substantially identical, each region comprisingat least eight different anchors (oligonucleotide, or one of the othertypes described herein), and

[0086] b) a container comprising at least one bifunctional linkermolecule, which has a first portion specific for at least one of saidanchor(s) and a second portion that comprises a probe which is specificfor at least one of said target(s).

[0087] In one embodiment, there is provided a surface as in a) above anda set of instructions for attaching to at least one of said anchors abifunctional linker molecule, which has a first portion specific for atleast one of said anchor(s) and a second portion that comprises a probewhich is specific for at least one target. The instructions can include,for example (but are not limited to), a description of each of theanchors on the surface, an indication of how many anchors there are andwhere on the surface they are located, and a protocol for specificallyattaching (associating, binding, etc.) the linkers to the anchors. Forexample, if the anchors are oligonucleotides, the instructions caninclude the sequence of each anchor, from which a practitioner candesign complementary anchor-specific moieties of linkers to interactspecifically with (e.g., hybridize to) the anchors; if the anchors arepeptides, the instructions can convey information about, e.g.,antibodies which will interact specifically with the peptides. Theinstructions can also include a protocol for associating the anchors andlinkers, e.g., conditions and reagents for hybridization (or other typeof association) such as temperature and time of incubation, conditionsand reagents for removing unassociated molecules (e.g., washes), and thelike. Furthermore, the instructions can include information on theconstruction and use of any of the types of control linkers discussedherein, and of methods, e.g., to quantitate, normalize, “fine-tune” orcalibrate assays to be performed with the combinations. The instructionscan encompass any of the parameters, conditions or embodiments disclosedin this application, all of which can be performed routinely, withconventional procedures, by one of skill in the art.

[0088] As discussed elsewhere in this application, a practitioner canattach to a surface of the invention comprising a given array (orarrays) of anchors, a wide variety of types of linkers, therebyprogramming any of a wide variety of probe arrays. Moreover, apractitioner can remove a given set of linkers from a surface of theinvention and add to it another set of linkers (either the same ordifferent from the first set), allowing a given surface to be reusedmany times. This flexibility and reusability constitute furtheradvantages of the invention.

[0089] In another embodiment, combinations of the invention can be usedto map ESTs (Expressed Sequence Tags). That is, MAPS assays can be usedto determine which, if any, of a group of ESTs were generated fromdifferent (or partially overlapping) portions of the same gene(s), andwhich, if any, are unique. FIGS. 18, 19, 20 and 21 illustrate such anassay, in this example an assay to determine which, if any, of 16 ESTsare “linked” to a common gene. A first step of the assay (see FIG. 18)is to assemble arrays in which each of the ESTs to be mapped isrepresented by at least one oligonucleotide probe that corresponds toit. A number of arrays equal to (or greater than) the number of ESTs tobe mapped are distributed in separate regions (e.g., wells) of asurface; in the illustrated example, the surface of the combinationcomprises 16 wells, each of which contains an array of 16 differentEST-specific oligonucleotides, numbered 1-16. An oligonucleotide which“corresponds to” an EST (is “EST-specific”) is one that is sufficientlycomplementary to an EST such that, under selected stringenthybridization conditions, the oligonucleotide will hybridizespecifically to that EST, but not to other, unrelated ESTs. AnEST-corresponding oligonucleotide of this type can bind specifically(under optimal conditions) to the coding or non-coding strand of a cDNAsynthesized from the gene from which the EST was originally generated orto an mRNA synthesized from the gene from which the EST was originallygenerated. Factors to be considered in designing oligonucleotides, andhybridization parameters to be optimized in order to achieve specifichybridization, are discussed elsewhere in this application. In order toassemble the arrays, linker molecules are prepared, each of whichcomprises a moiety specific for one of the anchors of a generic arrayplus a moiety comprising an oligonucleotide probe that corresponds toone of the ESTs to be mapped; and the linkers are attached to anchors asdescribed elsewhere in this application. In a subsequent step, analiquot of a sample comprising a mixture of nucleic acids (e.g., mRNA orsingle stranded or denatured cDNA), which may contain sequences that arecomplementary to one or more of the oligonucleotide probes, is added toeach of the regions (wells) which comprises a probe array; the mixtureis then incubated under routinely determined optimal conditions, therebypermitting nucleic acid to bind to complementary probes. If several ofthe EST-specific probes are complementary to different portions of asingle nucleic acid, that nucleic acid will bind to each of the loci inthe array at which one of those probes is located.

[0090] In a subsequent step, a different detector oligonucleotide (inthe illustrated example, detectors #1 to 16) is added to each region(well) (see FIG. 19). A detector oligonucleotide is designed for each ofthe ESTs to be mapped. Each EST-specific detector corresponds to adifferent (at least partially non-overlapping) portion of the EST thandoes the probe oligonucleotide, so that the probe and the detectoroligonucleotides do not interfere with one another. Consider, forexample, the ESTs depicted in FIG. 21, which correspond to ESTs 1, 2 and6 of FIGS. 18-20. FIG. 21 indicates that ESTs #1 and #2 were bothobtained from gene X (they are “linked”), whereas EST #6 was obtainedfrom a different, unrelated gene. If aliquots of a sample containing amixture of mRNAs, including one generated from gene X, are incubatedwith the probe arrays shown in FIGS. 18-20, the gene X mRNA will, underoptimal conditions, hybridize at the loci with probes 1 and 2, but notat those with probe 6. (Of course, each mRNA must be added in molarexcess over the sum of the probes to which it can hybridize.) Ifdetector oligonucleotide 1 is added to region (well) 1, it willhybridize to the gene X mRNA which is bound at loci 1 and 2 of the probearray, but not at locus 6. Similarly, if detector oligonucleotide 2 isadded to another well—say, well #2—it will also bind at loci 1 and 2,but not 6. In this fashion, one can determine in a high throughputmanner which of the ESTs are linked, i.e. code for portions of the samegene, and which ESTs are unique. For the hypothetical example shown inFIG. 20, the first 3 ESTs encode portions of the same gene, the last 5ESTs encode portions of another gene, and the remaining ESTs appear notto be linked. Conditions of hybridization, optional wash steps, methodsof detection, and the like are discussed elsewhere in this applicationwith regard to other MAPS assays. In order to confirm the linkage dataobtained by the MAPS assay, one could perform PCR reactions using pairsof EST-specific oligonucleotide probes as sense and anti-sense primers.Every pair of linked ESTs should yield a PCR product. Note that thispairwise PCR test could be performed to determine linkage directlywithout using the Linkage MAPS assay; however, many reactions would berequired, and each EST primer would have to be synthesized as both senseand anti-sense strands. For the illustrated example, 180 such reactionswould be required.

[0091] In one aspect, the invention relates to a method of determiningwhich of a plurality of ESTs are complementary to a given nucleic acid,comprising,

[0092] a) incubating an immobilized array of oligonucleotide probes, atleast one of which corresponds to each of said ESTs, with a test samplewhich may contain said given nucleic acid, to obtain a hybridizationproduct between said oligonucleotide probes and said nucleic acid,

[0093] b) incubating said hybridization product with a detectoroligonucleotide, which corresponds to an EST to which one of saidoligonucleotide probes corresponds, but which is specific for adifferent portion of the EST than is said oligonucleotide probe, and

[0094] c) detecting which oligonucleotide probes of said array arelabeled by said detector oligonucleotide,

[0095] wherein said array of oligonucleotide probes is immobilized on aregion of a combination, wherein said combination comprises

[0096] 1) a surface comprising a number of spatially discrete,substantially identical, regions equal to the number of ESTs to bestudied, each region comprising

[0097] 2) a number of different anchors equal to the number of ESTs tobe studied, each anchor in association with

[0098] 3) a bifunctional linker which has a first portion that isspecific for the anchor, and a second portion that comprises anoligonucleotide probe which corresponds to at least one of said ESTs.

[0099] In another aspect, the invention relates to a method as above,wherein of said ESTs may be complementary to said nucleic acid, andwherein each of said ESTs comprises two different oligonucleotidesequences, the first of which defines an oligonucleotide probecorresponding to said EST, and the second of which defines a detectoroligonucleotide corresponding to said EST, comprising,

[0100] a) contacting a sample which comprises molecules of said nucleicacid with at least one region of a combination, wherein said regioncomprises an array of oligonucleotide probes, at least one of whichcorresponds to each of said ESTs,

[0101] b) incubating said sample with said region, thereby permittingmolecules of said nucleic acid to bind to said EST-correspondingoligonucleotide probes which are complementary to portions of saidnucleic acid,

[0102] c) incubating said region comprising molecules of said nucleicacid bound to one or more of said EST-corresponding oligonucleotideprobes with a detector oligonucleotide which corresponds to an EST towhich a given one of the oligonucleotide probes of said arraycorresponds, thereby binding detector oligonucleotides to nucleic acidmolecules which have bound to said given oligonucleotide probe or toother oligonucleotide probes which are complementary to said nucleicacid,

[0103] d) detecting the presence of said detector oligonucleotides,thereby identifying which EST-corresponding oligonucleotide probes ofsaid array are complementary to portions of a nucleic acid which bindsto said given oligonucleotide EST-corresponding probe, therebyidentifying which ESTs are complementary to said given nucleic acidwherein said array of oligonucleotide probes is immobilized on a regionof a combination, wherein said combination comprises

[0104] 1) a surface comprising a number of spatially discrete,substantially identical regions equal to the number of ESTs to bestudied, each region comprising

[0105] 2) a number of different anchors equal to the number of ESTs tobe studied, each anchor in association with

[0106] 3) a bifunctional linker which has a first portion that isspecific for the anchor, and a second portion that comprises anoligonucleotide probe which corresponds to at least one of said ESTs.

[0107] The components of an EST mapping assay can be assembled in anyorder. For example, the anchors, linkers and ESTs can be assembledsequentially; or linkers and ESTs, in the presence or absence ofreporters, can be assembled in solution and then added to the anchors.

[0108] In another aspect, the invention relates to a method ofdetermining which of a plurality of ESTs are complementary to a givennucleic acid, comprising,

[0109] a) incubating a collection of bifunctional oligonucleotide linkermolecules, each of which comprises a first portion which is a probe thatcorresponds to at least one of said ESTs, and a second portion which isspecific for an anchor oligonucleotide, with a test sample which maycontain said given nucleic acid, to obtain a first hybridization productbetween said oligonucleotide probes and said nucleic acid,

[0110] b) incubating said first hybridization product with animmobilized array of anchor oligonucleotides, wherein each anchoroligonucleotide corresponds to the anchor-specific portion of at leastone of said linker molecules, to form a second hybridization productcomprising said anchors, said oligonucleotide probes and said nucleicacid, and

[0111] c) incubating either said first or said second hybridizationproduct with a detector oligonucleotide, which corresponds to an EST towhich one of said oligonucleotide probes corresponds, but which isspecific for a different portion of the EST than is said oligonucleotideprobe, and

[0112] d) detecting which oligonucleotide probes of said array arelabeled by said detector oligonucleotide,

[0113] wherein said array of anchor oligonucleotides is immobilized on aregion of a combination, wherein said combination comprises

[0114] 1) a surface comprising a number of spatially discrete,substantially identical, regions equal to the number of ESTs to bestudied, each region comprising

[0115] 2) a number of different anchors equal to the number of ESTs tobe studied.

[0116] Of course, the above methods for mapping ESTs can be used to maptest sequences (e.g., polynucleotides) onto any nucleic acid ofinterest. For example, one can determine if two or more cloned DNAfragments or cDNAs map to the same genomic DNA. Such a procedure couldaid, for example, in the structural elucidation of long, complex genes.In a similar manner, one can determine if one or more spliced outsequences or coding sequences map to the same genomic DNA. Such adetermination could be used, for example, in a diagnostic test todistinguish between a normal and a disease condition which arecharacterized by differential splicing patterns. Many other applicationsof the mapping method will be evident to one of skill in the art.

[0117] In another aspect, the invention relates to a method ofdetermining which of a plurality of polynucleotides are complementary toa given nucleic acid,

[0118] wherein one or more of said polynucleotides may be complementaryto said nucleic acid, and wherein each of said polynucleotides comprisestwo different oligonucleotide sequences, the first of which defines anoligonucleotide probe corresponding to said polynucleotide, and thesecond of which defines a detector oligonucleotide corresponding to saidpolynucleotide, comprising,

[0119] a) contacting a sample which comprises molecules of said nucleicacid with at least one region of a combination, wherein said regioncomprises an array of oligonucleotide probes, at least one of whichcorresponds to each of said polynucleotides,

[0120] b) incubating said sample with said region, thereby permittingmolecules of said nucleic acid to bind to saidpolynucleotide-corresponding oligonucleotide probes which arecomplementary to portions of said nucleic acid,

[0121] c) incubating said region comprising molecules of said nucleicacid bound to one or more of said polynucleotide-correspondingoligonucleotide probes with a detector oligonucleotide which correspondsto a polynucleotide to which a given one of the oligonucleotide probesof said array corresponds, thereby binding detector oligonucleotides tonucleic acid molecules which have bound to said given oligonucleotideprobe or to other oligonucleotide probes which are complementary to saidnucleic acid,

[0122] d) detecting the presence of said detector oligonucleotides,thereby identifying which polynucleotide-corresponding oligonucleotideprobes of said array are complementary to portions of a nucleic acidwhich binds to said given oligonucleotide polynucleotide-correspondingprobe, thereby identifying which polynucleotides are complementary tosaid given nucleic acid,

[0123] wherein said array of oligonucleotide probes is immobilized on aregion of a combination, wherein said combination comprises

[0124] 1) a surface comprising a number of spatially discrete,substantially identical, regions equal to the number of polynucleotidesto be studied, each region comprising

[0125] 2) a number of different anchors equal to the number ofpolynucleotides to be studied, each anchor in association with

[0126] 3) a bifunctional linker which has a first portion that isspecific for the anchor, and a second portion that comprises anoligonucleotide probe which corresponds to at least one of saidpolynucleotides.

[0127] In another aspect of the invention, the above methods to map ESTsor other polynucleotides further comprise removing unbound portions ofthe sample between one or more of the steps.

[0128] In another embodiment of the invention, one or more RNA targetsof interest (e.g., mRNA, or other types of RNA) are converted into cDNAsby reverse transcriptase, and these cDNAs are then hybridized to a probearray. This type of assay is illustrated schematically in FIG. 8. RNAextracts (or purified mRNA) are prepared from cells or tissues asdescribed herein. Reverse transcriptase and oligonucleotide primerswhich are specific for the RNAs of interest are then added to the RNAsample, and, using art-recognized conditions and procedures, which canbe routinely determined and optimized, the first strands of cDNAs aregenerated. The term “specific” primer refers to one that is sufficientlycomplementary to an mRNA of interest to bind to it under selectedstringent hybridization conditions and be recognized by reversetranscriptase, but which does not bind to undesired nucleic acid (seeabove for a discussion of appropriate reaction conditions to achievespecific hybridization). Residual RNA—mRNAs which were not recognized bythe specific primers, and/or other types of contaminating RNAs in an RNAextract, such as tRNA or rRNA—can be removed by any of a variety ofribonucleases or by chemical procedures, such as treatment with alkali,leaving behind the single strand cDNA, which is subsequently placed incontact with a MAPS probe array. The use of reverse transcriptase inthis method minimizes the need for extensive handling of RNA, which canbe sensitive to degradation by nucleases and thus difficult to workwith. Furthermore, the additional specificity engendered by the specificreverse transcriptase primers imparts an added layer of specificity tothe assay.

[0129] Optionally, the cDNAs described above can be amplified beforehybridization to the probe array to increase the signal strength. Theoligonucleotide reverse transcriptase primers described above cancomprise, at their 5′ ends, sequences (which can be about 22-27nucleotides long) that specify initiation sites for an RNA polymerase(e.g., T7, T3 or SP2 polymerase, or the like). In the example shown inFIG. 8, a T7 promoter sequence has been added to the reversetranscriptase primer. The polymerase recognition site becomesincorporated into the cDNA and can then serve as a recognition site formultiple rounds of transcription by the appropriate RNA polymerase (invitro transcription, or IVT). Optionally, the mRNAs so generated can beamplified further, using PCR and appropriate primers, or the cDNA,itself, can be so amplified. Procedures for transcription and PCR areroutine and well-known in the art.

[0130] The flexibility of PCR allows for many variations in the methodsof the invention. In one embodiment, one or both of the PCR primerswhich are used to amplify a target can comprise a chemical modificationwhich allows the resulting PCR product to attach, specifically ornon-specifically, to a solid support. Such chemical modificationsinclude, for example, 5′ amidation which allows binding to surfaces suchas Costar's DNA Bind Plates, (e.g., which are modified withN-oxysuccinimide ester, or maleic anhydride coated plates such asReacti-Bind plates from Pierce, Rockford, Ill.). Methods for generatingoligonucleotides comprising such chemical modifications are routine andconventional in the art. A PCR product comprising such a modified primercan be attached to any desired support, including a solid support, e.g.,the inner walls of a microtiter well, a bead (e.g., a non-magnetic ormagnetic bead), or any of the types of surfaces described herein. Ofcourse, a PCR primer can also be attached to a support before a PCRreaction is initiated. Several cycles of PCR can be repeated withoutwashing but with an excess of bound primer, so that the resulting PCRproduct remains attached to the support. The attachment of an amplifiedtarget sequence to a support can facilitate the washing (orpurification) of the target, either before it is contacted with (e.g.,hybridized to) a surface comprising anchors and/or linkers, or after ithas been contacted with and then released from such a surface.

[0131] In another embodiment, one or both of the PCR primers used toamplify a target can comprise one or more restriction enzyme sites,allowing the introduction of restriction sites adjacent to either endof, or flanking, a target sequence of interest. Restriction sites can beadded to an amplified target by PCR either before or after it hascontacted (e.g., hybridized to) a surface comprising anchors and/orlinkers. Restriction site(s) introduced in this manner can, for example,facilitate the cloning of an amplified target by providing cloning siteswhich flank the target sequence. Restriction sites can also facilitatethe purification of an amplified sequence. For example, one or morerestriction sites can be placed in a PCR primer between a targetspecific sequence and a chemical modification which allows attachment toa support. After a target has been PCR amplified, using the modified PCRprimer, and has bound to a support via the chemical modification, it canbe washed and then cleaved at the restriction site(s) adjacent to thetarget sequence, thereby releasing the washed target. See, e.g., FIG.23.

[0132] Of course, cleavable sites other than restriction enzyme sitescan also be used in the methods described above, e.g., a peptide whichcan be cleaved by a specific protease, or another component which can becleaved and/or released by physical, chemical or other means.

[0133] In another embodiment, one or both of the PCR primers used toamplify a target can comprise a sequence (which is not necessarilypresent in the target) that is specific for, e.g., a target-specificreporter or a detection linker.

[0134] Of course, the above-described primer modifications can be usedtogether in any desired combination, and can be added to an amplifiedproduct at any stage of an assay. Examples 21 and 22 demonstrateprotocols in which several of the primer modifications described aboveare incorporated into an amplified target.

[0135] The above-described methods, in which mRNA targets are convertedto cDNA with reverse transcriptase and/or are amplified by PCR beforeassaying on MAPS plates, can be used instead of the standard MAPS assayprocedure for any of the RNA-based assays described above.

[0136] Nucleic acids used in the methods of the invention, e.g.,targets, oligonucleotides involved in the detection of a target, ornuclease protection fragments (described elsewhere herein) can beamplified by any of a variety of conventional enzymatic procedures,including PCR and ligase reactions. One such amplification method isTranscription-Mediated Amplification (see, e.g., Abe et al. (1993). J.Clin. Microbiol. 31, 3270-3274). See also Example 32 and FIGS. 36-39 and42.

[0137] In another embodiment of the invention, one or more nucleic acidtargets of interest are hybridized to specific polynucleotide protectionfragments and subjected to a nuclease protection procedure, and thoseprotection fragments which have hybridized to the target(s) of interestare assayed on MAPS plates. Of course, such “MAPS plates” can containanchors which are not associated with linkers (e.g., which can beassociated directly with a target or nuclease protection fragment ofinterest); the advantages of nuclease protection as used in conjunctionwith any type of probe array will be evident to one of skill in the artfrom this specification and any of its ancestors to which benefit isclaimed. If the target of interest is an RNA and the protection fragmentis DNA, a Nuclease Protection/MAPS Assay (NPA-MAPS) can reduce the needfor extensive handling of RNA, which can be sensitive to degradation bycontaminating nucleases and thus difficult to work with. Treatment of asample with a nuclease protection procedure also allows for a samplewith reduced viscosity. Nuclease protection of a sample can allow forgreater sensitivity and reproducibility in an assay. See, e.g., Example30, which illustrates the sensitivity and reproducibility of a typicalassay in which a sample is treated with a nuclease protection procedure.An advantage of the invention is that assays can be sensitive enoughthat amplification of the target (e.g., by PCR) is not necessary inorder to detect a signal. In an NPA-MAPS assay, the probes in the probearray are oligonucleotides of the same strandedness as the nucleic acidtargets of interest, rather than being complementary to them, as in astandard MAPS assay. One example of an NPA-MAPS assay is schematicallyrepresented in FIG. 9.

[0138] In an NPA-MAPS assay, the target of interest can be any nucleicacid, e.g., genomic DNA, cDNA, viral DNA or RNA, rRNA, tRNA, mRNA,oligonucleotides, nucleic acid fragments, modified nucleic acids,synthetic nucleic acids, or the like. In a preferred embodiment of theinvention, the procedure is used to assay for one or more mRNA targetswhich are present in a tissue or cellular RNA extract. A sample whichcontains the target(s) of interest is first hybridized under selectedstringent conditions (see above for a discussion of appropriate reactionconditions to achieve specific hybridization) to an excess of one ormore specific protection fragment(s). A protection fragment is apolynucleotide, which can be, e.g., RNA, DNA (including a PCR product),PNA or modified or substituted nucleic acid, that is specific for aportion of a nucleic acid target of interest. By “specific” protectionfragment, it is meant a polynucleotide which is sufficientlycomplementary to its intended binding partner to bind to it underselected stringent conditions, but which will not bind to other,unintended nucleic acids. A protection fragment can be at least 10nucleotides in length, preferably 50 to about 100, or about as long as afull length cDNA. In a preferred embodiment, the protection fragmentsare single stranded DNA oligonucleotides. Protection fragments specificfor as many as 100 targets or more can be included in a singlehybridization reaction. After hybridization, the sample is treated witha cocktail of one or more nucleases so as to destroy nucleic acid otherthan the protection fragment(s) which have hybridized to the nucleicacid(s) of interest and (optionally) the portion(s) of nucleic acidtarget which have hybridized and been protected from nuclease digestionduring the nuclease protection procedure (are in a duplexed hybrid). Forexample, if the sample comprises a cellular extract, unwanted nucleicacids, such as genomic DNA, tRNA, rRNA and mRNA's other than those ofinterest, can be substantially destroyed in this step. Any of a varietyof nucleases can be used, including, e.g., pancreatic RNAse, mung beannuclease, S1 nuclease, RNAse A, Ribonuclease T1, Exonuclease III,Exonuclease VII, RNAse CLB, RNAse PhyM, RNAse U2, or the like, dependingon the nature of the hybridized complexes and of the undesirable nucleicacids present in the sample. RNAse H can be particularly useful fordigesting residual RNA bound to a DNA protection fragment. Reactionconditions for these enzymes are well-known in the art and can beoptimized empirically. Also, chemical procedures can be used, e.g.,alkali hydrolysis of RNA. As required, the samples can be treatedfurther by well-known procedures in the art to remove unhybridizedmaterial and/or to inactivate or remove residual enzymes (e.g., phenolextraction, precipitation, column filtration, etc.). The process ofhybridization, followed by nuclease digestion and (optionally) chemicaldegradation, is called a nuclease protection procedure; a variety ofnuclease protection procedures have been described (see, e.g., Lee et al(1987). Meth. Enzymol. 152, 633-648. Zinn et al (1983). Cell 34,865-879.). Samples treated by nuclease protection, followed by an(optional) procedure to inactivate nucleases, are placed in contact witha MAPS probe array and the usual steps of a MAPS assay are carried out.Bound protection fragments can be detected by, e.g., hybridization tolabeled target-specific reporters, as described herein for standard MAPSassays, or the protection fragments, themselves, can be labeled,covalently or non-covalently, with a detectable molecule.

[0139] If desired, one or more controls can be included for normalizingan NPA-MAPS assay. For example, one or more protection fragmentscorresponding to a nucleic acid which is expected to be present in eachof a series of samples in a substantially constant amount (e.g., aconstitutively produced mRNA, a portion of a genomic DNA, a tRNA orrRNA) can be used. The ability to detect and quantify an internalnormalization control, e.g., genomic DNA, in an assay for measuringnucleic acids which are present in variable amounts (e.g., mRNAs), is anadvantage of using protection fragments in the assays.

[0140] Because the amount of the normalization standard(s) may be lowerthan that of expressed mRNAs of interest, the assay may be adjusted sothe signals corresponding to the expressed genes do not swamp out thesignal(s) corresponding to the normalization standard(s). Methods ofadjusting the signal levels are conventional and will be evident to oneof skill in the art. For example, any of the methods described hereinfor balancing signal intensities (e.g., signal attenuation, fine-tuning)can be used (e.g., using blocked linkers; labeling the signal moietydesigned to detect the normalization standard at a greater level thanthat designed to detect the mRNA; placing at a locus designated fordetecting a normalization standard a plurality of linkers which arespecific for different portions of the normalization nucleic acid, orfor protection fragments that correspond to different portions of thatnucleic acid, etc.). The normalization standard(s) and the nucleic acidtargets (e.g., mRNAs) of interest can be detected simultaneously orsequentially, e.g., by any of the methods described elsewhere herein.Example 28 and FIG. 29 illustrate a typical experiment in which internalDNA normalization standards are used in an assay of mRNAs.

[0141] In a preferred embodiment, the protection fragment is directlylabeled, e.g., rather than being labeled by hybridization to atarget-specific reporter. For example, the reporter is bound to theprotection fragment through a ligand-antiligand interaction, e.g., astreptavidin enzyme complex is added to a biotinylated protectionoligonucleotide. In another example, the protection fragment is modifiedchemically, (e.g., by direct coupling of horseradish peroxidase (HRP) orof a fluorescent dye) and this chemical modification is detected, eitherwith the nucleic acid portion of the protection fragment or without it,(e.g., after cleavage of the modification by, for example, an enzymaticor chemical treatment). In any of the above methods, a protectionfragment can be labeled before or after it has hybridized to acorresponding linker molecule.

[0142] In order to control that the nuclease protection procedure hasworked properly, i.e. that non-hybridized nucleic acids have beendigested as desired, one can design one or more protection fragments tocontain overhanging (non-hybridizing) segments that should be cleaved bythe nucleases if the procedure works properly. The presence or absenceof the overhanging fragments can be determined by hybridization with acomplementary, labeled, detection probe, or the overhanging portion ofthe protection fragment, itself, can be labeled, covalently ornon-covalently, with a detectable molecule. This control can beperformed before the sample is placed in contact with the probe array,or as a part of the MAPS assay, itself. An example of such a controlassay is described in Example 15. Of course, because different labelscan be easily distinguished (e.g., fluors with different absorptionspectra), several differently labeled oligonucleotides can be includedin a single assay. Further, the standard nuclease protection assay asanalyzed by gel electrophoresis can be used during assay development toverify that the protection fragments are processed as expected.

[0143] Other controls for correct nuclease digestion will be evident toone of skill in the art. For example, one can include in an assay anuclease protection fragment which is known not to have specificity forany nucleic acid in the sample (e.g., in an assay for plant nucleicacids, one can include a protection fragment specific for an animal genewhich is known to be absent in plants).

[0144] After detection of targets, the detection probe (e.g.,HRP-labeled) signal can be eliminated (e.g. denatured, killed, quenched,suppressed, blocked), plates washed to remove any resulting reagents,agents, or buffers which might interfere in the next step (e.g.,denaturing regent), and then the overhang can be detected with adifferent detection probe (e.g., also HRP-labeled). The use of signaldenaturation followed by addition of a different detection probe withthe same signaling moiety can be used at various stages of the assay.Utilization of two different flourescent probes and dual color detectioncan be used without denaturation or signal blocking.

[0145] In one embodiment of the invention, as was noted above, anoligonucleotide probe is used to screen for a nucleic acid whichcomprises one or more polymorphisms. In a preferred embodiment, thenucleic acid (e.g., a DNA, such as a genomic DNA, or an RNA, such as anmRNA) comprises one or more SNPs. Routine, art-recognized procedures canbe used to carry out the procedure. For example, to screen for a DNAcomprising a known SNP, or an mRNA expressed from such a DNA, a“SNP-specific” protection fragment is hybridized to a sample comprisingnucleic acids which may comprise that SNP. By “SNP-specific” protectionfragment is meant in this context a protection fragment which comprisesthe altered base of the SNP or, if an mRNA is to be analyzed, thereverse complement of such a sequence. The sample is then treated withone or more appropriate nucleases which, under appropriate, empiricallydeterminable conditions, digest unhybridized single stranded nucleicacid and cleave double stranded (duplex) nucleic acid (e.g., DNA-DNAhybrids, DNA-RNA hybrids, or the like) at the site of a mismatch (e.g.,a single base mismatch). Appropriate nucleases include, e.g., SI orRHAse H. If a nucleic acid which comprises a SNP is present in thesample and hybridizes to the SNP-specific protection fragment, theprotection fragment will survive the digestion procedure intact, and canbe subjected to a MAPS assay and detected by a detection probe ordetection oligonucleotide which is specific for a sequence of theprotection fragment. Nucleic acids which do not comprise the SNP will becleaved at the site of the mismatch between the SNP-specific protectionfragment and the corresponding wild type sequence in the nucleic acid.If desired, a portion of the protection fragment which lies eitherdistal to or proximal to the site of cleavage can be removed, usingconventional methods (e.g., heat denaturation, enzymatic cleavage, etc.)An assay can be designed either so that the cleaved molecules (orportions thereof) will not bind to linkers, or so that such cleavedmolecules, even if a portion thereof binds to a linker, will not bedetected by an appropriately designed detection probe or detectionoligonucleotide. Example 29 and FIGS. 30 and 31 illustrate, i.a., thatassays of the invention can detect a single base mismatch in anexpressed SNP. Example 32 (FIG. 41) illustrates an assay for thedetection of SNPs, which is applicable, e.g., to the detection of SNPsin genomic DNA.

[0146] NPA-MAPS assays can be used to quantitate the amount of a targetin a sample. If protection fragment is added at a large enough molarexcess over the target to drive the hybridization reaction tocompletion, the amount of protection fragment remaining after thenuclease protection step will reflect how much target was present in thesample. One example of such a quantitation reaction is described inExamples 12 and 13.

[0147] In one embodiment of the invention, different types of targets ina sample, e.g., various combinations of DNA, RNA, intracellular proteinsand secreted proteins, can be assayed with a single probe array. SeeFIGS. 40 and 41 for examples of such assays.

[0148] NPA-MAPS assays can be used to implement any of the methodsdescribed above that use standard MAPS assays.

[0149] In a preferred embodiment, the polynucleotide protectionfragments are measured by the mass spectrometer rather than on MAPSplates. In a most preferred embodiment, none of the polynucleotides arebound (attached) to a solid surface during the hybridization or nucleasedigestion steps. After hybridization, the hybridized target can bedegraded, e.g., by nucleases or by chemical treatments, leaving theprotection fragment in direct proportion to how much fragment had beenhybridized to target. Alternatively, the sample can be treated so as toleave the (single strand) hybridized portion of the target, or theduplex formed by the hybridized target and the protection fragment, tobe further analyzed. The samples to be analyzed are separated from therest of the hybridization and nuclease mixture (for example by ethanolprecipitation or by adsorption or affinity chromatography, etc.), elutedor solubilized, and injected into the mass spectrometer by loopinjection for high throughput. In a preferred embodiment, the samples tobe analyzed (e.g., protection fragments) are adsorbed to a surface andanalyzed by laser desorption, using well-known methods in the art. Forhighest sensitivity Fourier Transform Mass Spectrometry (FTMS) (or othersimilar advanced technique) may be used, so that femtomoles or less ofeach protection fragment can be detected.

[0150] The protection fragments that are to be detected within one (ormore) samples can be designed to give a unique signal for the massspectrometer used. In one embodiment, the protection fragments each havea unique molecular weight after hybridization and nuclease treatment,and their molecular weights and characteristic ionization andfragmentation pattern will be sufficient to measure their concentration.To gain more sensitivity or to help in the analysis of complex mixtures,the protection fragments can be modified (e.g., derivatized) withchemical moieties designed to give clear unique signals. For exampleeach protection fragment can be derivatized with a different natural orunnatural amino acid attached through an amide bond to theoligonucleotide strand at one or more positions along the hybridizingportion of the strand. With a mass spectrometer of appropriate energy,fragmentation occurs at the amide bonds, releasing a characteristicproportion of the amino acids. This kind of approach in which chemicalmoieties of moderate size (roughly 80 to 200 molecular weight) are usedas mass spectrometric tags is desirable, because molecules of this sizeare generally easier to detect. In another example, the chemicalmodification is an organic molecule with a defined mass spectrometricsignal, such as a tetraalkylammonium group which can, for example,derivatize another molecule such as, e.g., an amino acid. In anotherexample, positive or negative ion signals are enhanced by reaction withany of a number of agents. For example, to enhance positive iondetection, one can react a pyrylium salt (such as, e.g., 2-4-dithenyl,6-ethyl pyrylium tetrafluoroborate, or many others) with an amine toform a pyridinium salt; any of a number of other enhancing agents can beused to form other positively charged functional groups (see, e.g.,Quirke et al (1994). Analytical Chemistry 66, 1302-1315). Similarly, onecan react any of a number of art-recognized agents to form negative ionenhancing species. The chemical modification can be detected, of course,either after having been cleaved from the nucleic acid, or while inassociation with the nucleic acid. By allowing each protection fragmentto be identified in a distinguishable manner, it is possible to assay(e.g., to screen) for a large number of different targets (e.g., for 2,6, 10, 16 or more different targets) in a single assay. Many such assayscan be performed rapidly and easily. Such an assay or set of assays canbe conducted, therefore, with high throughput as defined herein.

[0151] Regardless of whether oligonucleotides are detected directly bytheir mass or if unique molecular tags are used, the signals for eachmolecule to be detected can be fully characterized in pure preparationsof known concentration. This will allow for the signal to be quantified(measured, quantitated) accurately. For any molecule to be detected bymass spectrometry, the intensity and profile cannot be predicted withaccuracy. The tendency of the molecule to be ionized, the sensitivity ofall chemical bonds within the molecule to fragmentation, the degree towhich each fragment is multiply charged or singly charged, are all toocomplex to be predicted. However, for a given instrument with fixedenergy and sample handling characteristics the intensity and profile ofthe signal is very reproducible. Hence for each probe the signal can becharacterized with pure standards, and the experimental signalsinterpreted quantitatively with accuracy.

[0152] In one aspect, the invention relates to a method to detect one ormore nucleic acids of interest, comprising subjecting a samplecomprising the nucleic acid(s) of interest to nuclease protection withone or more protection fragments, and detecting the hybridized duplexmolecules, or the protected nucleic acid, or the protection fragment,with mass spectrometry.

[0153] Methods of analyzing nucleic acids by mass spectrometry arewell-known in the art. See, e.g., Alper et al (1998). Science 279,2044-2045 and Koster, U.S. Pat. No. 5,605,798.

[0154] In addition to the variety of high throughput assays describedabove, many others will be evident to one of skill in the art.

[0155] An advantage of using multiprobe assays is the ability to includea number of “control” probes in each probe array which are subject tothe same reaction conditions as the actual experimental probes. Forexample, each region in the array can comprise positive and/or negativecontrols. The term, a “positive control probe,” is used herein to mean acontrol probe that is known, e.g., to interact substantially with thetarget, or to interact with it in a quantitatively or qualitativelyknown manner, thereby acting as a(n internal) standard for theprobe/target interaction. Such a probe can control for hybridizationefficiency, for example. The term, a “negative control probe,” is usedherein to mean a control probe which is known not to interactsubstantially with the target. Such a probe can control forhybridization specificity, for example. As examples of the types ofcontrols which can be employed, consider an assay in which an array ofoligonucleotide probes is used to screen for agents which modulate theexpression of a set of correlative genes for a disease. As an internalnormalization control for variables such as the number of cells lysedfor each sample, the recovery of mRNA, or the hybridization efficiency,a probe array can comprise probes which are specific for one or morebasal level or constitutive house-keeping genes, such as structuralgenes (e.g., actin, tubulin, or others) or DNA binding proteins (e.g.,transcription regulation factors, or others), whose expression is notexpected to be modulated by the agents being tested. Furthermore, todetermine whether the agents being tested result in undesired sideeffects, such as cell death or toxicity, a probe array can compriseprobes which are specific for genes that are known to be induced as partof the apoptosis (programmed cell death) process, or which are inducedunder conditions of cell trauma (e.g., heat shock proteins) or celltoxicity (e.g., p450 genes).

[0156] Other control probes can be included in an array to “fine tune”the sensitivity of an assay. For example, consider an assay for an agentwhich modulates the production of mRNAs associated with a particulardisease state. If previous analyses have indicated that one of thecorrelative mRNAs (say, mRNA-A) in this set is produced in such highamounts compared to the others that its signal swamps out the othermRNAs, the linkers can be adjusted to “fine tune” the assay so as toequalize the strengths of the signals. “Blocked linkers,” which comprisethe anchor-specific oligonucleotide sequence designated for the mRNA-Atarget, but which lack the probe-specific sequence, can be added todilute the pool of target-specific linkers and thus to reduce thesensitivity of the assay to that mRNA. The appropriate ratios of blockedand unblocked linkers can be determined with routine, conventionalmethods by one of skill in the art.

[0157] The “fine tuning” of an assay for a particular target by dilutingan active element with an inactive element can also be done at othersteps in the assay. For example, it can be done at the level ofdetection by diluting a labeled, target-specific reporter with an“inactive” target-specific reporter, e.g., one with the sametarget-specific moiety (e.g., an oligonucleotide sequence) but without asignaling entity, or with an inactivated or inactive form of thesignaling entity. The term “signaling entity,” as used herein, refers toa label, tag, molecule, or any substance which emits a detectable signalor is capable of generating such a signal, e.g., a fluorescent molecule,luminescence enzyme, or any of the variety of signaling entities whichare disclosed herein). In an especially preferred embodiment, the “finetuning” can be done at the step of contacting a target-containingcomplex with a detection linker (detection linkers are described below,e.g., in the section concerning complex sandwich-type detection methods,Example 23, and FIG. 24). A set of detection linkers can be designed,e.g., to fine tune the sensitivity for each individual target in anassay. For example, if a particular target is known to be present in asample at very high levels, the detection linker for that target can bediluted with an empirically-determinable amount of “blocked detectionlinker,” comprising the target-specific moiety (e.g., oligonucleotidesequence) but no moiety specific for a reporter reagent, or comprisingthe target-specific moiety and a reporter reagent-specific moiety whichis pre-bound to an inactive reporter reagent. That is, instead ofcomprising a moiety specific for a reporter reagent, that moiety can beabsent, or prevented (e.g., blocked) from interacting with (e.g.,hybridizing to) the reporter reagent. Such fine tuning is sometimesreferred to herein as signal “attenuation.” FIG. 28 illustrates anexperiment in which such signal attenuation was performed.

[0158] Samples to be tested in an assay of the invention can compriseany of the targets described above, or others. Liquid samples to beassayed can be of any volume appropriate to the size of the test region,ranging from about 100 nanoliters to about 100 microliters. In apreferred embodiment, liquid drops of about 1 microliter are applied toeach well of a 1536 well microtiter dish. Samples can be placed incontact with the probe arrays by any of a variety of methods suitablefor high throughput analysis, e.g., by pipetting, inkjet baseddispensing or by use of a replicating pin tool. Samples are incubatedunder conditions (e.g., salt concentration, pH, temperature, time ofincubation, etc.—see above) effective for achieving binding or otherstable interaction of the probe and the target. These conditions areroutinely determinable. After incubation, the samples can optionally betreated (e.g., washed) to remove unbound target, using conditions whichare determined empirically to leave specific interactions intact, but toremove non-specifically bound material. For example, samples can bewashed between about one and ten times or more under the same orsomewhat more stringent conditions than those used to achieve theprobe/target binding.

[0159] Samples containing target RNA, e.g., mRNA, rRNA, tRNA, viral RNAor total RNA, can be prepared by any of a variety of procedures. Forexample, in vitro cell cultures from which mRNA is to be extracted canbe plated on the regions of a surface, such as in individual wells of amicrotiter plate. Optionally, these cells, after attaining a desiredcell density, can be treated with an agent of interest, such as astimulating agent or a potential therapeutic agent, which can be addedto the cells by any of a variety of means, e.g., with a replicating pintool (such as the 96 or 384 pin tools available from Beckman), bypipetting or by ink-jet dispensing, and incubated with the cells for anyappropriate time period, e.g., between about 15 minutes and about 48hours, depending upon the assay. Total RNA, mRNA, etc. extracts fromtissues or cells from an in vitro or in vivo source can be preparedusing routine, art-recognized methods (e.g., commercially availablekits).

[0160] In one embodiment, cells are lysed (or permeabilized), in thepresence or absence of nuclease protection fragment(s), and the crudelysate is used directly (e.g., in the well of a microtiter plate),without further purification from, e.g., other cellular components. Ifthe cells are lysed in the absence of nuclease protection fragments,such protection fragments can optionally be added subsequently to thelysate.

[0161] In a preferred embodiment, e.g., in which nuclease protectionfragments are detected, samples are prepared by contacting cells ofinterest (e.g., cells on the surface of a well of a microtiter plate;cells in a tissue or whole organism sample; or the like) with an aqueousmedium (lysis solution) which comprises a surfactant or detergent (e.g.,SDS, e.g., at about 0.01% to about 0.5% w/v) and an agent (e.g.,formamide (e.g., at about 8-about 60%, v/v), guanidium HCl (e.g., atabout 0.1-about 6M), guanidium isothyocyanate (e.g., at about 0.05-about8M) or urea (e.g., at about 40-about 46%, w/v, or about 7M)), which,alone or in combination with one or more other agents, can act as achaotropic agent. The aqueous medium can be buffered by any standardbuffer. In a preferred embodiment, the buffer is about 0.5-6×SSC, morepreferably about 3×SSC. Optionally, the aqueous medium can also comprisetRNA at an appropriate concentration, e.g., about 0.1-2.0 mg/ml,preferably about 0.5 mg/ml. Nuclease protection fragments may also beadded to the aqueous medium before it is added to the cells. The optimalconcentration of each protection fragment can be determined empirically,using conventional methods. In a preferred embodiment, the concentrationof each protection fragment is about 3 to about 300 pM, more preferablyabout 30 pM.

[0162] Cells are incubated in the aqueous solution until the cellsbecome permeabilized and/or lysed, and DNA and/or mRNA is released fromthe cells into the aqueous medium. Cells are incubated in the aqueousmedium for an empirically determinable period of time (e.g., about 1 minto about 60 min), at an empirically determinable optimizable temperature(e.g., about 37° C. to about 115° C., preferably about 90° C. to about115° C.)

[0163] For example, in one embodiment, in which both DNA and RNA arereleased from the cells in a denatured form capable of binding to aprotection fragment, the cells are incubated for about 1 min to about 60min, preferably about 5 to about 20 min, in the aqueous medium at about90° to about 115° C., preferably about 105° C. If desired, e.g., when itis desirable to assay for DNA in the absence of RNA, any of a variety ofconventional ribonucleases can be included in the incubation mixture.Selection of an appropriate ribonuclease, and optimization of digestionconditions, are conventional and readily determined by a skilled worker.

[0164] In another embodiment, mRNA can be prepared by incubating cellsfor about 5 to about 20 min, preferably about 10 min, in an aqueousmedium at about 90° to about 100° C., preferably about 95° C.,optionally in the presence of one or more protection fragments. In thiscase, mRNA is substantially released from the cells in a denatured formcapable of binding to a protection fragment, and DNA remainssubstantially inside or attached to the cells, or is unavailable to aprobe by virtue of its double-stranded nature, or is released from thecells, but in a form which is not able to bind to a protection fragment(e.g., is not denatured). Without wishing to be bound to any particularmechanism, it appears that, as the nucleic acid is released from thelysed/permeabilized cells, it is sufficiently denatured to allow it tobind to a protection fragment to form a stable duplex which is resistantto degradation by endogenous or exogenous reagents or enzymes, andproteins within the cell (e.g., nucleases) are denatured and/orinactivated.

[0165] Following preparation of a nucleic acid of interest by the aboveprocedure, the sample can be diluted, in the appropriate volume, so thatthe aqueous medium does not inhibit the function of exogenously addedproteins such as, e.g., nucleases (e.g., S1 nuclease), polymerases(e.g., polymerases required for PCR reactions), or binding proteins(e.g., streptavidin). The amounts of dilution, and the identity andamounts of the components to be used in the aqueous solution, asdescribed above, can be determined empirically, using conventionalmethods.

[0166] For any of the methods of this invention, targets can be labeled(tagged) by any of a variety of procedures which are well-known in theart and/or which are described elsewhere herein (e.g., for the detectionof nuclease protection fragments). For example, the target molecules canbe coupled directly or indirectly with chemical groups that provide asignal for detection, such as chemiluminescent molecules, or enzymeswhich catalyze the production of chemiluminsecent molecules, orfluorescent molecules like fluorescein or cy5, or a time resolvedfluorescent molecule like one of the chelated lanthanide metals, or aradioactive compound. Alternatively, the targets can be labeled afterthey have reacted with the probe by one or more labeled target-specificreporters (e.g., antibodies, oligonucleotides as shown in FIG. 1, or anyof the general types of molecules discussed above in conjunction withprobes and targets).

[0167] One type of fluorescent molecule can be an “upconvertingphosphore,” i.e., a fluor which absorbs and is excited at a longwavelength (e.g, IR), then emits at a shorter wavelength (e.g., visiblelight). Because upconverting phosphores absorb at a longer wavelengththan do most potentially interfering materials present in a typicalsample to be analyzed, upconverting phosphores allow a reduction ininterference caused by material in the sample, compared to phosphoreswhich absorb at a lower wavelength. The narrow emission spectrum of mostupconverting phosphores also allows the simultaneous detection of alarge number of different upconverting phosphores. Upconvertingphosphores are well-known and conventional in the art, and include,e.g., rare earth metal ions such as, e.g., Ytterbium (Yb), Erbium (Er),Thulium (Tm) and Praseodymium (Pr), particularly in the form of anoxysulfide salt. As many as 80 or more independently detectableupconverting phosphores have been described. (See, e.g., BiologicalAgent Detection and Identification, Apr. 27-30, 1999, DARPA, BiologicalWarfare Defense, Defense Sciences Office. The phosphores can optionallybe attached to any surface, e.g., to a microsphere or a latex bead. Likeother fluorescent labels, upconverting phosphores can be detected byenergy transfer to (or modulation by) the label on a sufficiently closelinker, target or reporter. Furthermore, as with other signalingentities disclosed herein, upconverting phosphores can be used toquantitate the amount of a target, and can be used in any of the varietyof procedures described herein, e.g., to detect nuclease protectionfragments.

[0168] Of course, upconverting phosphores can also be used to detecttargets which are distributed in any other fashion on a surface, e.g.,targets (including nuclease protection fragments) which are bounddirectly to a surface, bound directly to an array of differentoligonucleotides on a surface, or bound via bifunctional linkers toanchors (different or substantially identical) which are distributedsubstantially evenly, or in any desired organization or pattern, on asurface. Any surface can be used, e.g., a flow-through system, or asolid surface such as, e.g., a bead. Beads used in any of the assays ofthe invention can be of any type, e.g., made of any material, magneticand/or non-magnetic; and the beads used in a single assay can be ofsubstantially the same, or different, sizes and/or shapes.

[0169] A variety of more complex sandwich-type detection procedures canalso be employed. For example, a target can be hybridized to abifunctional molecule containing a first moiety which is specific forthe target and a second moiety which can be recognized by a common(i.e., the same) reporter reagent, e.g., a labeled polynucleotide,antibody or the like. The bifunctional molecules can be designed so thatany desired number of common reporters can be used in each assay.

[0170] For any of the methods of this invention, a variety of complexsandwich-type detection procedures can be employed to label (tag)targets. For example, a target can interact with, e.g., hybridize to, abifunctional (or multifunctional) molecule (a “detection linker”)containing a first moiety that is specific for the target and a secondmoiety that is specific for a “reporter reagent.” The term “specificfor” has the meaning as used herein with respect to the interactions of,e.g., probes and targets. The term “reporter reagent,” as used herein,refers to a labeled polynucleotide, antibody or any of the general typesof molecules discussed herein in conjunction with probes and targets.These two moieties of a detection linker can recognize (interact orassociate with) their respective binding partners in any of the mannersdiscussed above in conjunction, e.g., with probes and targets. Adetection linker can also comprise other sequences, e.g., sequences thatare specific for a target but are different from (non-overlapping with)the target-specific moiety of the corresponding anchor-bound linker. Anysequence present in a detection linker can serve as a recognitionsequence for a detection probe or a reporter agent. In a preferredembodiment, a detection linker is a polynucleotide.

[0171] Detection linkers can be designed so that any desired number ofcommon reporter reagents can be used in an assay. For example, a set ofdetection linkers can be designed such that each detection linker isspecific for a different target, but comprises a binding site for thesame (common), or for one of a limited number of, reporter reagents. Theability to use a limited number of (e.g., one) reporter reagents tolabel a variety of targets in a single assay provides the advantage ofreduced cost and lower backgrounds. Of course, detection linker/reporterreagent combinations can be used to detect targets which are distributedin any fashion on a surface, e.g., as described above for the types oftarget arrangements that can be detected by upconverting phosphores.

[0172] In a most preferred embodiment, detection linkers can be designedto detect nuclease protection fragments in such a way that protectionfragments which have been cleaved by a nuclease from control “overhang”sequences during a nuclease protection procedure (as described, e.g., inExample 15) are preferentially labeled. This type of detection procedureis illustrated schematically in FIG. 24. In this embodiment, a detectionlinker comprises a first moiety that is specific for a target and asecond moiety that is specific for the common control overhang sequencewhich, in a preferred embodiment, is present on substantially all of thenuclease protection fragments at the start of an assay. If, as desired,the control overhang sequence has been cleaved from a nucleaseprotection fragment during a nuclease protection reaction, thetarget-specific moiety of the detection linker will hybridize to thecleaved protection fragment, but the control overhang-specific moiety ofthe detection linker will be unbound and will remain available forfurther hybridization. If, on the other hand, the controloverhang-specific sequence is not cleaved from a protection fragment,e.g., because of incomplete nuclease digestion during a nucleaseprotection procedure, both the target-specific and the controloverhang-specific moieties of the detection linker will hybridize to theprotection fragment and will not be available for further hybridization.In a preferred embodiment, complexes comprising nuclease protectionfragments and bound detection linkers are then hybridized in a furtherstep to a reporter reagent which comprises a signaling entity (e.g., afluorochrome, hapten, enzyme, or any other molecule bearing a detectablesignal or signal-generating entity, as described herein) and an moiety(e.g., an oligonucleotide) which is specific for the controloverhang-specific moiety of a detection linker. The reporter reagentwill preferentially bind to and label those complexes in which thecontrol overhang sequence of the nuclease protection fragment has beencleaved off, (i.e., a complex in which the control overhang-specificmoiety of the detection linker is available for further hybridization tothe reporter reagent.)

[0173] Numerous other variations of sandwich detection procedures willbe evident to one of skill in the art.

[0174] Methods by which targets can be incubated with a target-specificreporter(s), or target/detection linker complexes can be incubated withreporter reagents, under conditions effective for achieving binding orother stable interaction, are routinely determinable (see above). Forexample, fluorescent oligonucleotide reporters (at a concentration ofabout 10 nM to about 1 μM or more, preferably about 30 nM, in a buffersuch as 6×SSPE-T or others) can be incubated with the bound targets forbetween about 15 minutes to 2 hours or more (preferably about 30 to 60minutes), at a temperature between about 15° C. and about 45° C.(preferably about room temperature). After incubation, the samples canoptionally be treated (e.g., washed) to remove unbound target-specificreporters, using conditions which are determined empirically to leavespecific interactions intact, but to remove non-specifically boundmaterial. For example, samples can be washed between about one and tentimes or more under the same or somewhat more stringent conditions thanthose used to achieve the target/reporter binding.

[0175] Tagging with a target-specific reporter(s) can provide anadditional layer of specificity to the initial hybridization reaction,e.g., in the case in which a target-specific oligonucleotide reporter istargeted to a different portion of the sequence of a target nucleic acidthan is the probe oligonucleotide, or in which probe and reporterantibodies recognize different epitopes of a target antigen.Furthermore, tagging with target-specific reporters can allow for“tuning” the sensitivity of the reaction. For example, if a target mRNAwhich is part of a correlative expression pattern is expressed at verylow levels, the level of signal can be enhanced (signal amplification)by hybridizing the bound target to several (e.g., about two to aboutfive or more) target-specific oligonucleotide reporters, each of whichhybridizes specifically to a different portion of the target mRNA.

[0176] The ability to detect two types of labels independently allowsfor an additional type of control in MAPS assays. Some (e.g., about 10to about 100%) of the linkers designated for a particular anchor locus(FIG. 7 shows 3 typical anchor loci, each comprising a plurality ofsubstantially identical anchors (A, B or C)) can have a label (e.g., afluor) attached to one end. For example, a rhodamine or Cy5 fluor can beattached at the 5′ end of the linker. Such modified linkers are termed“control linkers.” After a mixture of linkers and control linkers hasbeen associated with anchors and a sample containing a target has beenincubated with the resulting probe array, a target-specific reporterbearing a different fluor (e.g., fluorescein or another detection labelsuch as a chemiluminescent one) can be used (or the target can bedirectly labeled with a fluor or other detection label); and the ratioof the two signals can be determined. The presence of control linkerspermits calibration of the number of functional (e.g., able to interactwith linkers) anchors within and between test regions (i.e. tests thecapacity of each locus of the array to bind target, for purposes ofnormalizing signals), serves as a basis for quantitation of the amountof bound target, aids in localization of the anchor loci and/or providesa positive control, e.g., in cases in which there is no signal as aresult of absence of target in a sample. In one embodiment of theinvention, two different labels (e.g., fluorophores) can also be used todetect two different populations of target molecules; however, theability to recognize the presence of targets by spatial resolution ofsignals allows the use of a single type of label for different targetmolecules.

[0177] The ability to detect labels independently (e.g., fluorescentlabels which emit at distinguishable wavelengths, such as, e.g.,fluorescein and rhodamine, or different upconverting phosphores) allowsadditional flexibility in the methods of the invention. For example,each of two or more targets can be labeled, directly or indirectly, withits own, uniquely detectable, label. This allows for the detection oftargets on the basis of features specific to the labels (e.g., color ofthe emission) in addition to (or instead of), e.g., identifying theposition of a localized target on a surface, or identifying a target byvirtue of the size of a bead to which it is localized. In anotherembodiment of the invention, a multiplicity of targets can be detectedindependently at a single locus within a region. For example, two ormore (e.g., 2, 3, 4, 5, 6 or more) targets can be detected at a locuswhich is defined by a single group of (substantially identical) anchors.That is, a set of linkers can be used, each of which has ananchor-specific portion specific for the same anchor plus atarget-specific portion specific for a different target. If a set of,e.g., four such linkers is used, all four can bind to members of thegroup of anchors at a single locus, allowing four different targets tobind at that locus. If each of these targets is labeled (directly orindirectly) with a different, distinguishable, label, an investigatorcan determine the presence of each of the four targets at the locusindependently. Therefore, an array of, e.g., five anchors (groups ofanchors) in a region can be used in the scenario described above todetect as many as twenty different targets. Such an assay is illustratedin Example 24 and FIG. 25. Similarly, a plurality of targets, e.g., asmany as 80 or more, can be detected independently when a single type ofanchor is distributed, not at a single locus, but evenly, or in anydesired fashion, on a solid surface such as, e.g., a bead or a flowthrough apparatus; and other aspects such as bead size or scatter can beused to provide information about target identity or groups of targets.

[0178] The association of multiple linkers (e.g., ranging from about twoto about 50 or more), having different target specificities, with theanchors at a given locus (either a group of substantially identicalanchors or a “mixed locus”), sometimes referred to herein as “mixedlinkers,” forms the basis for other embodiments of the invention, whichwill be evident to those of skill in the art. For example, at a givenlocus the anchors can be bound to a mixture of linkers which arespecific for a plurality of different protection fragments, each ofwhich corresponds to (is specific for) a different portion of a nucleicacid (e.g., an mRNA) of interest. The presence of such a plurality ofdifferent linkers at a locus allows for considerably increasedsensitivity in the detection of a target (e.g., an mRNA) of interest,e.g., one present at low abundance in a sample. Each locus can bedesigned so that the number of linkers corresponding to differentportions of an mRNA designated for that locus is inversely proportional(in an empirically determinable fashion) to the abundance of that mRNAin the sample. For example, if one mRNA of interest is found in apreliminary experiment to be present in a sample in large excess over asecond mRNA of interest, the relative number of linkers corresponding todifferent portions of the two mRNAs can be adjusted so that the relativeintensities of the signals corresponding to each mRNA are substantiallythe same. That is, the signal intensities can be adjusted so that thesignal corresponding to the first mRNA does not swamp out the signalcorresponding to the second mRNA. In this manner, one can adjust anassay to allow for simultaneous detection of a plurality of mRNAs whichare present in widely different amounts in a sample, balancing thesignal intensity corresponding to each mRNA.

[0179] In another embodiment of the invention, as was noted above, agiven locus can comprise linkers which are specific for a plurality ofunrelated or different targets or protection fragments, allowing for thedetection of a greatly increased number of targets or protectionfragments with a single array of anchors. If, for example, each locus ofan array of 350 anchors comprises linkers specific for 10 differenttargets, then the array can be used to detect as many as 3500 targets.In effect, such an arrangement allows one to convert an array which candetect a low density of targets to one which can detect a high densityof targets.

[0180] Multiple molecules (e.g., protection fragments) bound at a singlelocus can be detected sequentially or simultaneously, e.g., using thedetection methods described elsewhere in this application. (For adiscussion of “detection linkers” and “reporter reagents,” see, e.g.,the section above concerning complex sandwich-type detection methods.)In one embodiment, a first target (e.g., a protection fragment) at agiven locus is detected, e.g., with a first detection system (e.g., adetection linker/reporter reagent, or a detection probe specific forit); then that first detection linker/reporter reagent or probe isremoved or inactivated, using conventional procedures (e.g., changingthe pH to inactivate a reporter reagent comprising an enzyme thatgenerates a chemoluminescent signal), and a second detectionlinker/reporter reagent or detection probe specific for a second targetat the same locus is used to detect that second target; and so forth foras many cycles as desired. In another embodiment, the first detectionlinker/reporter reagent or detection probe is added to a combination asabove, but it is not removed or inactivated before the second detectionlinker/reporter reagent or detection probe is added. In this embodiment,the amount of signal corresponding to the second target can bedetermined by subtracting out the amount of signal corresponding to thefirst target. In another embodiment, the first and second detectionlinker/reporter reagents or detection probes are added to thecombinations as above, substantially simultaneously, and are detectedindividually, e.g., using differentially detectable labels as describedelsewhere herein. In any of the detection methods described herein, thedetection linkers can comprise moieties which are specific for the sameor for different reporter reagents. For example, if four targets areassociated with the linkers at a given locus, the detection linkersspecific for each of the four targets can each comprise a moietyspecific for a different reporter reagent. Therefore, after the set ofall four detection linkers is hybridized to the targets, the targets canbe detected sequentially or simultaneously, as described above, usingthe four different reporter reagents. Other detection methods, as wellas combinations of the above methods, will be evident to one of skill inthe art.

[0181] Of course, “mixed linkers” are also advantageous for use withsurfaces which contain a single (non-repeated) region.

[0182] In another embodiment of the invention, “anchors” which arespecific for a target(s) of interest are not associated with linkers,but rather are associated directly with the target(s); the target(s), inturn, can interact optionally with detection linker(s) or with detectionprobe(s).

[0183] Targets, whether labeled or unlabeled, can be detected by any ofa variety of procedures, which are routine and conventional in the art(see, e.g., Fodor et al (1996). U.S. Pat. No. 5,510,270; Pirrung et al(1992). U.S. Pat. No. 5,143,854; Koster (1997). U.S. Pat. No. 5,605,798;Hollis et al (1997) U.S. Pat. No. 5,653,939; Heller (1996). U.S. Pat.No. 5,565,322; Eggers et al (1997). U.S. Pat. No. 5,670,322; Lipshutz etal (1995). BioTechniques 19, 442-447; Southern (1996). Trends inGenetics 12, 110-115). Detection methods include enzyme-based detection,calorimetric methods, SPA, autoradiography, mass spectrometry,electrical methods, detection of absorbance or luminescence (includingchemiluminescence or electroluminescence), and detection of lightscatter from, e.g., microscopic particles used as tags. Also,fluorescent labels can be detected, e.g., by imaging with acharge-coupled device (CCD) or fluorescence microscopy (e.g., scanningor confocal fluorescence microscopy), or by coupling a scanning systemwith a CCD array or photomultiplier tube, or by using array-basedtechnology for detection (e.g., surface potential of each 10-micron partof a test region can be detected or surface plasmon resonance can beused if resolution can be made high enough.) Alternatively, an array cancontain a label (e.g., one of a pair of energy transfer probes, such asfluorescein and rhodamine) which can be detected by energy transfer to(or modulation by) the label on a linker, target or reporter. Among thehost of fluorescence-based detection systems are fluorescence intensity,fluorescence polarization (FP), time-resolved fluorescence, fluorescenceresonance energy transfer and homogeneous time-released fluorescence(HTRF). Analysis of repeating bar-code-like patterns can be accomplishedby pattern recognition (finding the appropriate spot or line for eachspecific labeled target by its position relative to the other spots orlines) followed by quantification of the intensity of the labels.Bar-code recognition devices and computer software for the analysis ofone or two dimensional arrays are routinely generated and/orcommercially available (e.g., see Rava et al (1996). U.S. Pat. No.5,545,531).

[0184] Another method which can be used for detection is 2-photonfluorescence, including applications where the fluorescence ofendogenous or conjugated fluorochromes of components bound to the arraysurface is enhanced by being bound close to the surface of the array,for instance by close apposition to the substrate on which the array isformed, or by close apposition to other agents included in the anchor orlinker or otherwise incorporated in the bound complex. Otherfluorescence methods or utility include lifetime fluorescence,polarization, energy transfer, etc. For instance, such methods permitthe simultaneous detection and descrimination of multiple targets withinthe same locus, and in some instances can discriminate between boundlabel and unbound label, eliminating the need to wash unbound lable awayfrom the array, and thus facilitating the measurment of rapidlyreversible or weak interactions by the array.

[0185] Methods of making and using the arrays of this invention,including preparing surfaces or regions such as those described herein,synthesizing or purifying and attaching or assembling substances such asthose of the anchors, linkers, probes and detector probes describedherein, and detecting and analyzing labeled or tagged substances asdescribed herein, are well known and conventional technology. Inaddition to methods disclosed in the references cited above, see, e.g.,patents assigned to Affymax, Affymetrix, Nanogen, Protogene, Spectragen,Millipore and Beckman (from whom products useful for the invention areavailable); standard textbooks of molecular biology and protein science,including those cited above; and Cozette et al (1991). U.S. Pat. No.5,063,081; Southern (1996), Current Opinion in Biotechnology 7, 85-88;Chee et al (1996). Science 274, 610-614; and Fodor et al (1993). Nature364, 555-556.

BRIEF DESCRIPTION OF THE DRAWINGS

[0186]FIG. 1 illustrates a design scheme for oligonucleotides, in whicha linker 1 contains a portion that is specific for anchor 1 and anotherportion (a probe) that is specific for target mRNA 1, and in which alabeled detection probe 1 is specific for a sequence of target mRNA 1which is different from the sequence of the target-specific portion ofthe linker.

[0187]FIG. 2 illustrates a surface which comprises 15 test regions, eachof which comprises an array of six anchor oligonucleotides.

[0188]FIG. 3 illustrates the design of a linker for a receptor bindingassay, in which the anchor-specific portion of the linker is associatedwith the probe portion (the receptor protein) via biotin andstreptavidin molecules, and in which a ligand specific for the receptoris labeled with a fluorescent labeling molecule. B: Biotin. SA:Streptavidin. Rec: Receptor protein. Ligand: a natural or syntheticligand for the receptor. *: a fluorescent labeling molecule attached tothe Ligand.

[0189]FIG. 4 illustrates a surface which comprises 21 test regions, eachof which is further subdivided into 16 subregions (indentations,dimples).

[0190]FIGS. 5a, 5 b and 5 c illustrate three pieces from which a surfacesuch as that shown in FIG. 4 can be assembled. FIG. 5a represents a wellseparator; FIG. 5b represents a subdivider; and FIG. 5c represents abase.

[0191]FIG. 6 represents two test regions, each of which comprises alinear array of probes (or anchors) which are in a “bar-code”-likeformation.

[0192]FIG. 7 schematically represents a test region comprising 3 anchors(A, B and C), each of which is present in multiple copies (a “group”).The location of each group of anchors is termed a “locus.”

[0193]FIG. 8 illustrates an assay in which cDNA(s) generated by specificreverse transcriptase are assayed on MAPS plates.

[0194]FIG. 9 illustrates an assay which uses a nuclease protectionprocedure (NPA-MAPS assay). Sample RNA is prepared from cells or fromtissue and is represented as thin wavy lines. To the RNA sample is addeda group of polynucleotide protection fragments, portrayed as thick, darkand light lines. The dark sections of the protection fragments representsegments that are complementary to specific RNA targets and hybridize tothose targets. The light sections represent overhanging portions:sequences contiguous with the complementary sequence but notcomplementary to target. The protection fragments are added in excess.Following hybridization of all available target to the protectionfragments, the samples are treated with an appropriate cocktail ofnucleases and with chemical treatments that destroy unwantednon-hybridized RNA and non-hybridized polynucleotide. For example, S1nuclease can destroy any single stranded DNA present. Hence, excessprotection fragment is hydrolyzed as is the overhanging non-hybridizedportion of bound protection fragment. RNA can be hydrolyzed by additionof ribonucleases including ribonuclease H and or by heating samples inbase. Remaining is a collection of cleaved protection fragments thatreflect how much of each target RNA had been present in the sample. Theremaining protection fragments are measured by a MAPS hybridizationassay.

[0195]FIG. 10 illustrates hybridization specificity in a MAPS assay.

[0196]FIG. 11 illustrates binding kinetics of an anchor to a linker.

[0197]FIG. 12 illustrates a MAPS assay of two oligonucleotide targets.

[0198]FIG. 13 illustrates the quantification of a sensitivity shift.

[0199]FIG. 14 illustrates melting temperature determinations for fouroligonucleotide linker/anchor combinations.

[0200]FIG. 15 illustrates an mRNA assay by NPA-MAPS.

[0201]FIG. 16 illustrates a dilution curve with NPA-MAPS.

[0202]FIG. 17 illustrates an assay to detect peptides containingphosphotyrosine residues.

[0203]FIG. 18 illustrates the first step in an assay to map ESTs:assembling linkers corresponding to each of the ESTs to be mapped onarrays of generic anchors on a MAPS plate. To the surface of each of 16wells of a microplate are attached linkers comprising 16 differentoligonucleotide probes, arranged in a 4×4 matrix. The first locus hasoligo 1, which is complementary to a portion of the first EST sequence,and so on for the 16 ESTs to be tested.

[0204] cDNAs or mRNAs generated from the genes from which the ESTs wereobtained are added to all 16 wells and allowed to hybridize underappropriate conditions. Hence, any cDNA or mRNA that contains one of the16 EST sequences will be assembled at the locus where its complementaryprobe was placed.

[0205]FIG. 19 illustrates a subsequent step in an assay to map ESTs:adding detector oligonucleotides to the MAPS plate. Each well of theplate receives a detector oligonucleotide which corresponds to one ofthe ESTs to be mapped. Each detector oligonucleotide is anoligonucleotide coupled to a molecule used for detection, e.g.,fluorescein if fluorescence imaging is to be the method of detection.Each detector oligonucleotide is complementary to one of the ESTs, butdifferent from the EST-specific probe, so that a probe and a detectoroligonucleotide which are complementary to a single EST can both bind atthe same time.

[0206] After washing, a single detector oligonucleotide is added to eachwell, as numbered in the figure. That is, the detector oligonucleotidewith sequences complementary to the first EST is added to the firstwell, and so on.

[0207]FIG. 20a and b illustrate the results of the assay to map ESTsshown in FIGS. 18 and 19. After hybridization of detectoroligonucleotides and washing with appropriate conditions of stringency,the 16 wells of the microplate are imaged with a CCD-based fluorescenceimager, for example. FIG. 20a shows stylized results. It is expectedthat each EST-specific detector oligonucleotide should label the mRNA orcDNA held down by the corresponding EST-specific probe. For example,probe 5 assembles the cDNA or mRNA containing the fifth EST sequence atthat locus, so the fifth detector oligonucleotide should also hybridizeto the cDNA or mRNA at the same locus. This is the case for thesestylized data, with each detection oligonucleotide labeling the matchingprobe. In addition, the first three detector oligonucleotides each labelcDNA or mRNA held down by the first three probes, showing that thesesequences lie along the same gene. Similarly, the last five ESTs appearto be linked. The linkage assigned from these data are presentedgraphically in FIG. 20b.

[0208]FIG. 21 illustrates the relationships of the probes, detectoroligonucleotides and ESTs #1, 2 and 6 shown in FIGS. 18-20.

[0209]FIG. 22 illustrates a high throughput assay.

[0210]FIG. 23 illustrates a method to prepare an amplified target.

[0211]FIG. 24 illustrates an assay with detection linkers and reporteragents.

[0212]FIG. 25 illustrates a use of multiple flours.

[0213]FIG. 26 illustrates a high throughput assay.

[0214]FIG. 27 illustrates the spatial arrangement of genes for the THP-1cells, along with two sample cells of data (selected from FIG. 26).

[0215]FIG. 28 illustrates an assay with signal attenuation.

[0216]FIG. 29 illustrates an assay in which genomic DNA and expressedRNA are measured from the same sample in the same well, e.g., in whichgenomic DNA serves as a normalization control. The left panel depictsthe measurement of DNA alone; the right panel depicts the measurement ofboth the DNA and GAPDH RNA (measured in each corner of the array).

[0217]FIGS. 30 and 31 illustrate the detection of expressed SNPs.

[0218] FIGS. 32-35 illustrate the sensitivity and reproducibility of anassay.

[0219]FIG. 36 illustrates some types of assay configurations encompassedby the invention.

[0220]FIG. 37 illustrates nuclease protection fragment amplification byPCR.

[0221]FIG. 38 illustrates nuclease protection fragment amplification byLigase.

[0222]FIG. 39 illustrates nuclease protection fragment amplification byNuclease Protection.

[0223]FIGS. 40 and 41 illustrate assays in which, e.g., protein and mRNAare assayed together from the same sample.

[0224]FIG. 42 illustrates nuclease protection fragment amplification bypolymerase. An application for the detection of SNPs is illustrated.

EXAMPLES Example 1 Hybridization Specificity (see FIG. 10)

[0225] A generic MAPS plate was produced by using an inkjet dispenser,the Pixus system (Cartesian Technologies, Inc., Irvine, Calif.) to forman identical grid of DNA within each well of a microtiter plate. Alloligonucleotides were purchased from Biosource International (Camarillo,Calif.). For this plate, seven different oligonucleotide anchors weredispensed within each well in the pattern shown as the Key (left side ofthe figure). Each oligonucleotide was dispensed as a 10 nanoliterdroplet to two spots, from a 2 uM solution containing 500 mM sodiumphosphate pH 8.5 and 1 mM EDTA to the wells of a DNA Bind plate (ComingCostar), and allowed to dry. After attachment, wells were blocked with50 mM Tris pH 8, and then oligonucleotide that had not covalentlyattached to the surface was washed away with 0.1% SDS in 5×SSP buffer.

[0226] To the washed plate fluorescently labeled linker oligonucleotideswere added and allowed to hybridize in 6×SSPE with 0.1% Triton X-100 atroom temperature for thirty minutes. This is a preferred protocol forattachment of linkers. The linker oligonucleotides were cy5-derivatizedduring synthesis, and were complementary in 25 base-pair segments tospecific anchoring oligonucleotides. The sequences of the seven anchorsand linkers were as follows (all shown 5′ to 3′): #1 Anchor*:TCCACGTGAGGACCGGACGGCGTCC SEQ ID:1 Linker**GTCGTTTCCATCTTTGCAGTCATAGGATACTGAGTGGACGC SEQ ID:2 CGTCCGGTCCTCACGTGGARNA mimic(mouse C-jun): CTATGACTGCAAAGATGGAAACGACGATACTGAGTTGGACC SEQID:3 TAACATTCGATCTCATTCA Detector Oligonucleotide***TGAATGAGATCGAATGTTAGGTCCA SEQ ID:4 #2 Anchor*: CACTACGGCTGAGCACGTGCGCTGCSEQ ID:5 Linker** CTAGGCTGAAGTGTGGCTGGAGTCTGCAGCGCACGTGCTCA SEQ ID:6GCCGTAGTG RNA mimic (mouse MIP-2):AGACTCCAGCCACACTTCAGCCTAGGATACTGAGTCTGAAC SEQ ID:7 AAAGGCAAGGCTAACTGACDetector Oligonucloeotide*** GTCAGTTAGCCTTGCCTTTGTTCAG SEQ ID:8 #3Anchor*: GTCAGTTAGCCTTGCCTTTGTTCAG SEQ ID:9 Linker**ACCATGTAGTTGAGGTCAATGAAGGGCGCTCCCACAACGCT SEQ ID:10 CGACCGGCG RNA mimic(mouse GAPDH): CCTTCATTGACCTCAACTACATGGTGATACTGAGTGGAGAA SEQ ID:11ACCTGCCAAGTATGATGAC Detector Oligonucloeotide***GTCATCATACTTGGCAGGTTTCTCC SEQ ID:12 #4 Anchor*:GAACCGCTCGCGTGTTCTACAGCCA SEQ ID:13 Linker**CTACCGAGCAAACTGGAAATGAAATTGGCTGTAGAACACGC SEQ ID:14 GAGCGGTTC RNA mimic(mouse L32 protein): ATTTCATTTCCAGTTTGCTCGGTAGGATACTGAGTGAGTCA SEQ ID:15CCAATCCCAACGCCAGGCT Detector Oligonuc1oeotide***AGCCTGGCGTTGGGATTGGTGACTC SEQ ID:16 #5 Anchor*:CTCGTTCCGCGTCCGTGGCTGCCAG SEQ ID:17 Linker** CTGGCAGCCACGGACGCGGAACGAGSEQ ID:18 #6 Anchor*: CGGTCGGCATGGTACCACAGTCCGC SEQ ID:19 Linker**GCGGACTGTGGTACCATGCCGACCG SEQ ID:20 #7 Anchor*:GCGCGCCGCGTTATGCATCTCTTCG SEQ ID:21 Linker** CGAAGAGATGCATAACGCGGCGCCGSEQ ID:22

[0227] To each well either one linker or a mixture of linkers (asindicated in the figure) was added in bulk. (To the well marked “all”was added a mixture of all seven linkers.) Following incubation andwashing in 5×SSP 3 times, the fluorescence picture shown on the rightportion of the figure was taken with a Tundra imager (IRI, St.Catherines, Ontario). As can be seen, the linkers self-assembled to thesurface, by specifically associating with their complementary anchors.

[0228] This process is repeated except that eight different anchors aredispersed in each well and linkers subsequently preferentiallyassociated therewith. The entire process is repeated with 36, 64 etc.different anchors in each well of a 24, 96, 384, 864 or 1536 well plate.

Example 2 Binding Kinetics (see FIG. 11)

[0229] The rate of hybridization of Cy5-derivatized linker number 1 toits complementary attached anchor is shown, for different concentrationsof linker. The generic MAPS plate was prepared as for FIG. 1, exceptanchor 1 was attached at four spots per well. Incubations were done atroom temperature in 5×SSP with 0.1% tween-20, wells were washed 3 timeswith 5×SSP, and bound fluorescence was measured. A fluorescence pictureof the plate was taken with the Tundra, and background was subtractedand the integrated intensity of each spot within each well wascalculated with Tundra software. Plotted is the average and standarddeviation for the integrated intensity for the four spots within each oftwo duplicate wells.

Example 3 Fluorescent Linker

[0230] A generic MAPS plate is produced with one anchoringoligonucleotide spotted to either 1 spot per well (top two rows), 4spots per well (next four rows) or 16 spots per well (lower two rows),according to the methods discussed above. To each well complementary,fluorescently labeled, linker is attached by the preferred protocoldescribed in Example 1. Following washing the fluorescence picture ofthe plate is taken with the Tundra. The amount of fluorescence at eachspot reports how much functional linker is available to hybridize totarget. The amount of signal detected at repeated spots is highlyreproducible.

Example 4 Binding Curves

[0231] To the plate prepared as described in Example 3, is addeddifferent concentrations of a target oligonucleotide. The linker thathas been associated contains a 25-mer sequence complementary to aportion of the target. The target is added in 5×SSC with 0.05% SDS in atotal volume of either 30 or 100 microliters, and the plate is coveredand incubated at 50° C. overnight. Following hybridization of the targetto the attached linker, the target is visualized by a preferred protocolusing chemiluminescence. A biotinylated detector oligonucleotide,containing a 25-mer sequence complementary to a separate portion of thetarget (not to the same portion complementary to linker) is added at 30nM. Biotinylated detector can be added for 30 minutes after washing awayexcess unattached target, or it can be added along with target for thelength of the overnight hybridization. Following attachment of detector,the surface is washed twice with 5×SSC, once with 1×SSP containing 0.1%Tween-20 and 1% PEG (SSPTP), and a 1:50,000 dilution of 250 ug/ml HorseRadish Peroxidase conjugated to Streptavidin (HRP:SA, from Pierce,Rockford, Ill.) is added for 5 hours in SSPTP at room temperature. Wellsare washed four times with SSPTP, and washed once and then incubatedwith Super Signal Ultra reagent (Pierce). After a few minutes, picturesof luminescence are collected with the Tundra imager, e.g., the picturecan accumulate within the CCD array for five minutes. Low levels oftarget can be visualized in some wells at a target concentration of aslittle as ˜5×10⁻¹³ M; the amount of signal generally becomes saturatedat a target concentration of ˜10⁻¹⁰ M. The amount of signal detected atrepeated spots is highly reproducible.

Example 5 Assay of Two Oligonucleotides (see FIG. 12)

[0232] A binding curve demonstrating a MAPS hybridization assay usingthe preferred protocol discussed above for two different targetoligonucleotides is shown. A generic MAPS plate was prepared with fourdifferent anchoring oligonucleotides each spotted four times within eachwell. For the second and fourth anchor, complementary linkeroligonucleotides were self-assembled onto the surface as described. Twotargets were added at the concentrations shown in 40 microliters to eachwell as described, and incubated at 50° C. overnight. The amount of eachtarget attached was visualized by attaching biotinylated detectionoligonucleotide specific for each target followed by HRP:SA andchemiluminescence imaging as described. In the lower panel the intensityof the image is quantified. Software that is part of the Tundra Imagerpackage was used to scan the intensity of the images along lines betweenthe arrows shown in the upper panel. At the lowest concentration oftarget, 1.1 pM, the scanned images show well-defined gaussian peaks ateach spot, while there are no discernable background peaks seen in theleft-most sample, at 0 concentration of target.

Example 6 Sensitivity Shifting (see FIG. 13)

[0233] A MAPS hybridization assay can be used for measuring theconcentration of a set of oligonucleotides, by binding them to a surfaceand labeling them. This works well for those oligonucleotides which areat modest or low concentration. Two samples can be distinguished in sucha case because if one sample contains more oligonucleotide, more willbind. On the other hand, if the concentration of targetedoligonucleotide is saturating for the surface (i.e. if it is high enoughto occupy all binding sites), then if the concentration goes up no morecan bind, so the amount cannot be measured. However, the binding curveof a target can be shifted by adding unlabeled competing ligand.

[0234] Binding data are obtained for four different oligonucleotidetargets, all of which saturate the surface (i.e. reach maximal binding)at roughly 3 nM. By adding unlabeled competitive targets to all wells,the binding of labeled oligonucleotide is shifted, so that less binds atthe lower concentration, and the level at which saturation occurs ismoved up. One can add competitive oligonucleotides for, say, targets 1and 3 but not 2 and 4. This shifts the sensitivity of the assay only fortargets 1 and 3. In this way oligonucleotide targets of widely differentconcentrations can be measured within one assay well, if the relativeamount of oligonucleotide expected is known.

[0235] The data can be quantified as explained above for the binding ofone of the oligonucleotide targets. FIG. 13 shows quantitatively thatincluding competitive oligonucleotide in the assay shifts the bindingcurve used to assay for this target to higher concentrations.

Example 7 Melting Temperature of Four Probes (see FIG. 14)

[0236] The amount of four different fluorescent labeled linkeroligonucleotides specifically hybridized to anchor oligonucleotides bythe MAPS assay is plotted as the temperature is raised. The fouroligonucleotides were first allowed to hybridize at 50° C. for 1 hour at300 nM. Then the wells were washed with SSC without probes, and theamount bound was measured as above by fluorescence (50° C. point). Thenthe surface was incubated at 55° C. for 30 minutes and the fluorescencebound measured, and so on for all temperatures presented.

Example 8 Detection Methods

[0237] Two detection methods can be compared directly. To a MAPS platewith four oligonucleotide anchors attached, each at four spots per well,are added two oligonucleotides to each well, with both including acovalently attached cy5 moiety or both containing a biotin group. Theepi-fluorescence measurement is performed as described for viewing andmeasurement of the fluorescent linker. The chemiluminescencemeasurements are performed as described for the MAPS assay usingsubsequent addition of HRP:SA and a chemiluminescence substrate. Thesignals generated are roughly of the same magnitude. However, for thegeometry of the microplates, which contain walls separating each well,and occasional bubbles of liquid or a miniscus of fluid, reflections inthe epi-fluorescence images can cause interference in datainterpretation.

Example 9 Chemiluminescence Products

[0238] Two products available as chemiluminescence substrates for horseradish peroxidase can be compared as detection procedures for the MAPSassay. A MAPS plate is prepared as for Example 8, and incubated withbiotinylated linker oligonucleotides. Then either alkaline phosphatasecoupled to streptavidin (AlkPhos:SA) or HRP:SA is added, followed bywashing and addition of either CDP-Star (Tropix) to the wells withAlkPhos:SA or ECL-Plus to the wells with HRP:SA. Labeling with SAderivatized enzymes and substrates is as suggested by the manufacturersfor use in labeling of western blots. These two (as well as otheravailable substrates) can both be used to assess oligonucleotidehybridization to MAPS plates.

Example 10 Resolution at 0.6 mm

[0239] The resolution of the current system for MAPS assay is tested bypreparing a MAPS plate with four different oligonucleotide anchors perwell each spotted four times per well, with a pitch (center-to-centerspacing) of 0.6 mm. Then either cy5-derivatized linkers or biotinylatedlinkers are hybridized and detected and scanned as above. For theepi-fluorescence measurement the resolution is higher (and pitch couldlikely be reduced). For the chemiluminescence detection procedureneighboring spots are not completely separated, yet at this spacingindividual peaks may be resolved unambiguously by computerdeconvolution.

Example 11 Test Nuclease Protection Protocol

[0240] In an assay to test for the optimal conditions for hybridizationand nuclease treatment for the nuclease protection protocol, theNuclease Protection Assay kit from Ambion (Austin, Tex.) is used toprovide conditions, buffers and enzymes. Eight samples are prepared inone of three buffers. Hyb Buff 1 is 100% Hybridization Buffer (Ambion);Hyb Buff 2 is 75% Hybridization Buffer and 25% Hybridization DilutionBuffer (Ambion); and Hyb Buff 3 is 50% of each. A 70-mer oligonucleotidethat contains 60 residues complementary to a test mRNA is synthesized(Biosource International, Camarillo, Calif.) and labeled withPsoralen-fluorescein (Schleicher and Schuell, Keene, N H) following theprotocol as suggested for labeling of Psoralen-biotin by Ambion.Briefly, protection fragment is diluted to 50 ug/ml in 20 μls of TEbuffer(10 mM Tris, 1 mM EDTA, pH 8) boiled for 10 minutes, and rapidlycooled in ice water. Four μls of 130 ug/ml Psoralen-fluorescein in DMFis added, and the sample is illuminated for 45 minutes at 40° C. with ahand-held long wavelength UV source. Free Psoralen-fluorescein isremoved by extraction with saturated butanol. The mRNA used is GAPDHanti-sense mRNA, prepared from antisense plasmid (pTRI-GAPDH-Mouseantisense Control Template from Ambion) using T7 promoter and theMaxiScript kit (Ambion). The short protection fragment is the 60-mercomplementary portion synthesized separately and similarly labeled. Thesequences of the protection fragments are as follows: Full lengthprotection fragment: CGAGAAATATGACAACTCACTCAAGATTGTCAGCAATGCAT SEQ ID:23CCTGCACCACCAACTGCTTGCTTGTCTAA Short protection fragment:CGAGAAATATGACAACTCACTCAAGATTGTCAGCAATGCAT SEQ ID:24 CCTGCACCACCAACTGCTT

[0241] Hybridizations are done by mixing protection fragments at 20 nMand GAPDH mRNA at 60 nM in 10 μls final volume for two hours at 22° C.or 37° C. Following hybridization, 200 μls of a mixture of nucleases isadded according to instructions from the manufacturer (Ambion NucleaseProtection Kit, 1:200 dilution of nuclease mixture) and incubated againat the same temperatures for 30 minutes. Hydrolysis is stopped withHybridization Inhibition Buffer (Ambion), and oligonucleotides arepelleted and washed with Ethanol. 10 μls of 1×Gel Loading Buffer(Ambion) is added and oligonucleotides are separated on a 15% TBE-ureagel. The gel is swirled in running buffer for 30 minutes, put on aplastic plate and imaged with the Tundra using fluorescein filters forselecting excitation and emission wavelengths. The image is accumulatedon the CCD array for 2 minutes. Best conditions are those for samplesincubated in Hyb Buff 2 at 37° C. or in Hyb Buff 3 at 22° C. In thesesamples no detectable full-length protection fragment remains, andsignificant amounts of a portion of the full-length protection fragmentat a size apparently the same as the short protection fragment are seen.

Example 12 mRNA Assay by NPA-MAPS. (see FIG. 15)

[0242] The full NPA-MAPS protocol was used, with conditions forhybridization and nuclease treatment similar to those described inExample 11. Ten samples were run for the assay. All contained the sameamount of the 70-mer oligonucleotide protection fragment and differentamounts of GAPDH mRNA. Hybridization samples in 10 μls in 50%Hybridization Buffer and 50% Dilution Buffer containing 0.08 mg/ml YeastRNA (Ambion) were heated to 90° C. for 6 minutes, briefly centrifuged,heated to 70° C. for 5 minutes, and allowed to cool to 19° C. andincubated for 19 hours. 200 μls of nuclease mixture was then added toeach sample for 30 minutes at 19° C. 60 μls was aliquoted from eachsample for the MAPS assay. 2 μl of 10 N NaOH and 2 μl of 0.5 M EDTA wasadded, and the sample heated to 90° C. for 15 minutes, 37° C. for 15minutes, and allowed to sit at room temperature for 20 minutes. Thensamples were neutralized with 2 μl of 10 M HCl, and 12 μls of 20×SSCcontaining 2 M HEPES pH 7.5 and 200 nM biotinylated detectoroligonucleotide specific for the protection fragment was added alongwith 1 μl of 10% SDS. Samples were mixed, heated to 80° C. for 5minutes, and two 35 μl aliquots of each sample were pipetted to twowells of a MAPS plate (each sample was split in two and run in duplicateon the MAPS plate). The plate had been prepared as for standard MAPSprotocol, with self-assembled CY5-derivatized linker specific for theprotection fragment already attached. The MAPS plate was covered andincubated at 50° C. overnight, and detection and luminescence performedas described. In the last sample, no nucleases were added during theassay as a control to visualize how the protection fragment alone wouldbe detected by MAPS. In the lower portion of the figure, the intensityscan (as analyzed by the imager) for the top row of wells is presented.The amount of GAPDH mRNA present in the sample (that is, the amount ineach duplicate well after aliquoting to the MAPS plate) is listed in thefigure.

[0243] The oligonucleotides used for the MAPS plates were as follows:Anchor*: CGCCGGTCGAGCGTTGTGGGAGCGC SEQ ID:25 Linker**CTTGAGTGAGTTGTCATATTTCTCGGATACTGAGTGCGCTC SEQ ID:26 CCACAACGCTCGACCGGCGProtection fragment (complementary to mouse antisense mRNA for GAPDH)CGAGAAATATGACAACTCACTCAAGATTGTCAGCAATGCAT SEQ ID:27CCTGCACCACCAACTGCTTGCTTGTCTAA Detector Oligonucloeotide***- labeled at5′ end with biotin AAGCAGTTGGTGGTGCAGGATGCAT SEQ ID:28

Example 13 Dilution Curve, NPA-MAPS (see FIG. 16)

[0244] The data discussed in Example 12 and shown in FIG. 15 werequantified and plotted as a dilution curve. The average and standarddeviations for all eight spots of the two duplicate wells are plottedfor each concentration of mRNA. A binding curve is superimposed, of theform:

Fraction Bound=Max Bound*1/(1+IC ₅₀ /L)

[0245] where Max Bound is the maximum bound at saturation, FractionBound is the amount bound at ligand concentration, L, and the IC₅₀ isthe concentration of ligand at which the Fraction Bound is half of MaxBound. The curve is shown as red dots on the figure, drawn with a bestfit value of IC₅₀=4.2 femtomoles as labeled in the figure.

Example 14 NPA-MAPS Assay of GAPDH mRNA in a Total Mouse Liver RNAExtract

[0246] A total mouse RNA extract is assayed for GAPDH mRNA with anNPA-MAPS assay and a dilution curve is made. Total RNA from mouse liveris prepared using a Qiagen kit. RNA is precipitated in 70% EtOH with 0.5M Mg-Acetate, and resuspended in 10 μls of 5×SSC with 0.05% SDS with 1.8nM protection fragment. The protection fragment added is anoligonucleotide 70 bases long, 60 bases of which are complementary tomouse GAPDH. Either a fragment complementary to mouse GAPDH mRNA is used(“protection fragment”), or the complement of the sequence is used as anegative control

[0247] (“antisense fragment”).

[0248] RNA samples with protection fragments are heated to 90° C. for 5minutes, and hybridizations are done by bringing samples to 70° C. andallowing them to cool slowly to room temperature over night. S1 nuclease(Promega) at 1:10 dilution is added in 30 μls of 1×S1 Nuclease Buffer(Promega) for 30 minutes at 19° C., and stopped by 1.6 μls of 10 N NaOHand 2.7 μls of 0.5 M EDTA. Samples are heated to 90° C. for 15 minutesand then 37° C. for 15 minutes to denature and destroy RNA, neutralizedwith 1.6 μls of 10 M HCl, and incubated on MAPS plates overnight in5×SSC with 0.05% SDS supplemented with 200 mM HEPES pH 7.5 to which 30nM biotinylated detection oligonucleotide is added. Washing andvisualization with SA-HRP is done as described. The amount of signaldecreases in parallel with decreasing amounts of mouse RNA (samplesinclude 500, 170, 50, 5, or 0.5 μg of total mouse RNA. Two controlsamples are included to which no S1 nuclease is added. Signal is seenonly for the complementary protection fragment.

[0249] Oligonucleotides used: For Antisense Control (sameoligonucleotides as for example 12): Anchor*: CGCCGGTCGAGCGTTGTGGGAGCGCSEQ ID:25 Linker** CTTGAGTGAGTTGTCATATTTCTCGGATACTGAGTGCGCTC SEQ ID:26CCACAACGCTCGACCGGCG Protection fragment (complementary to mouseantisense mRNA for GAPDH) CGAGAAATATGACAACTCACTCAAGATTGTCAGCAATGCAT SEQID:27 CCTGCACCACCAACTGCTTGCTTGTCTAA Detector Oligonucloeotide***AAGCAGTTGGTGGTGCAGGATGCAT SEQ ID:28 For Sense GAPDH mRNA samples:Anchor*: CGCCGGTCGAGCGTTGTGGGAGCGC SEQ ID:25 Linker**ATGCATCCTGCACCACCAACTGCTTGATACTGAGTGCGCTC SEQ ID:29 CCACAACGCTCGACCGGCGProtection fragment (complementary to mouse mRINA for GAPDH):AAGCAGTTGGTGGTGCAGGATGCATTGCTGACAATCTTGAG SEQ ID:30TGAGTTGTCATATTTCTCGGCTTGTCTAA Detector Oligonucleotide***CGAGAAATATGACAACTCACTCAAG SEQ ID:31

Example 15 A Nuclease Protection MAPS Assay with Controls

[0250] mRNA is extracted from mouse liver and nuclease protection isperformed essentially as described in Example 14, except that the GADPHspecific protection fragment comprises 60 nucleotides which arecomplementary to mouse GAPDH, followed by 15 “overhanging” nucleotidesat the 3′ end of the fragment which are not complementary to the target.After hybridization and nuclease digestion the remaining protectionfragment is hybridized to a MAPS plate as indicated in Example 14,except that two different oligonucleotide detection fragments are usedto detect the immobilized protection fragment. One detection fragment iscomplementary to the GAPDH-specific portion of the protection fragment,and the other, a control, is complementary to the 15 base overhangportion of the protection fragment. Each detection fragment is used ondifferent replicate samples (i.e., in different wells), so that bothdetection fragments can be labeled with the same detection molecule. Inthe present example both fragments are labeled with HRP. Without theaddition of nuclease, signals from both of the detection fragments areseen; whereas, when nuclease digestion is performed only the signalcorresponding to the GAPDH sequences can be detected. The amount ofGAPDH-specific signal is reduced relative to that observed in theabsence of nuclease digestion, because the protection fragment is addedat excess relative to the amount of GAPDH mRNA present. This allows theamount of GAPDH mRNA to be limiting to the protective hybridization, sothat the amount of double-stranded hybrid formed (and therefore theamount of protection fragment that is protected from the nuclease)reflects the amount of mRNA. When no mRNA is included in the reactionmixture, neither signal can be detected when nucleases are added. Theabove findings demonstrate that the hybridization and digestion steps ofthe assay occurred as desired.

[0251] When protection fragments corresponding to a variety of targetsare included in a given assay, each of the protection fragments cancomprise the same 15 base overhang portion. This allows for onedetection fragment to be used to test for remaining overhang for allsamples.

Example 16 A Transcription Assay Screening for Compounds that May Alterthe Expression of Genes that are Correlative with a Disease State

[0252] A cell line derived from a human tumor is used. It is found toexpress 30 genes at higher levels than do normal cells. (That is, these30 genes are being used more than in normal cells, to make mRNA and thento make the protein for which the genes are the instructions. Atranscription assay measures how much the genes are being used bymeasuring how much mRNA for each gene is present.) Using a nucleaseprotection assay on MAPS plates (NPA-MAPS), 8800 chemical compounds aretested to see if growing the cells in the presence of the compounds canreduce the expression of some of the 30 correlative genes withoutaffecting the expression of six normal (constitutive, “housekeeping”)genes. Any compounds having that effect might be useful in the futuredevelopment of drugs for treating this kind of tumor.

[0253] About 10,000 to 100,000 cells are added to each well of 10096-well polystyrene plates and the cells are grown for 2 days until theycover the surface of each well. For 8 wells of each plate, the cells areleft to grow without additions. To the remaining 88 wells of each plate,a different chemical compound is added so that the effect of it alonecan be tested. For the 100 plates used at one time, 8800 compounds canbe tested or screened. The cells are grown for 24 hours in the presenceof the compounds, and then the cells are harvested for assay. The cellsin each plate are treated according to the instructions for preparingRNA in samples from 96-well plates (for example according to the QiagenRNeasy 96 kit). After the RNA is prepared, the amount of each of 36different mRNA species is quantified by the NPA-MAPS approach, includingthe 30 correlative genes and 6 normal “housekeeping” genes. 36 DNAoligonucleotide protection fragments, each corresponding to one of thegenes of interest, are added to each well and allowed to hybridize underselected stringent conditions to their target mRNA sequences. Then S1nuclease is added to destroy excess unhybridized DNA, and the samplesare treated chemically to destroy the RNA as well. Left is theoligonucleotide protection fragment for each of the 36 genes inproportion to how much mRNA had been present in the treated cells foreach sample.

[0254] One hundred 96-well plates, each of which comprises an array of aplurality of 36 different anchor oligonucleotides in each well, areprepared by adding to each well 36 different linker oligonucleotides.The linkers self-assemble on the surface of each well, converting thegeneric plates to MAPS plates comprising specific probes for each of the36 oligonucleotide protection fragments. Each linker has a portionspecific for one of the 36 anchors and a portion specific for a segmentof one of the 36 protection oligonucleotides. The oligonucleotide samplefrom each well of the 100 sample plates is added to a corresponding wellof the 100 MAPS plates. After hybridization under selected stringentconditions, a detection oligonucleotide for each target with achemiluminescent enzyme attached is added, so that each specific spot ofeach well lights up in proportion to how much mRNA had been present inthe sample. Any wells that show reduced amounts of correlative geneswith no effect on the 6 house keeping genes are interesting. Thecompounds added to the cells for those samples are possible startingpoints to develop anti-tumor agents.

Example 17 Induced and Constitutive Gene Expression

[0255] RNA was prepared essentially as described in Example 14, from thelivers of mice either not infected (“Control”) or one hour afterinfection (“Infected”) by adenovirus. 60 μgs of liver RNA was used foreach sample, and samples were prepared in duplicate. Each assay wellcontained three sets of duplicate loci, corresponding to the three genesdescribed above. Each locus contained an anchor, bound to a linkercomprising a probe which was complementary to a protection fragmentcorresponding to one of the three genes. A nuclease protection MAPSassay was performed essentially as described in FIG. 12, and the imageswere collected and scanned as described. Shown are the raw image datacollected and the intensity scans for duplicate wells for each of thethree mRNA targets. The numbers over the scan lines are the integratedintensity values and standard deviations for each condition (n=4). Thehouse-keeping gene, GAPDH, not expected to change, showed a modestincrease of 1.3-fold in the infected sample that was not statisticallysignificant. The transcription of MIP-2 and c-jun was increased 4 and6-fold, respectively. These findings demonstrate that two genes, MIP-2and c-jun, exhibit enhanced expression in response to adenovirusinfection, compared to a control, constitutively expressed gene—GAPDH.

Example 18 An Enzyme Assay Screening for Compounds that SelectivelyInhibit Tyrosine or Serine Kinases (see FIG. 17).

[0256] Kinases are enzymes that attach a phosphate to proteins. Manyhave been shown to stimulate normal and neoplastic cell growth. Hence,compounds that inhibit specific kinases (but not all kinases) can beused to test whether the kinases are involved in pathology and, if so,to serve as starting points for pharmaceutical development. For example,five tyrosine kinases that are involved in stimulating cell growth or inregulating the inflammatory response are src, Ick, fyn, Zap70, and yes.Each kinase has substrates that are partially identified, as shortpeptides that contain a tyrosine. Some of the kinase specificitiesoverlap so that different kinases may phosphorylate some peptidesequally but others preferentially. For the five kinases, 36 peptidesubstrates are selected that show a spectrum of specific and overlappingspecificities.

[0257] One hundred 96-well plates are used; each well comprises 36generic oligonucleotide anchors. 36 linkers are prepared to convert thegeneric oligonucleotide array (with anchors only) to arrays comprisingpeptide substrates. The 36 peptide substrates are synthesized and eachis attached covalently through an amide bond, for example, to anoligonucleotide containing a 5′ amino group. The oligonucleotidescontain sequences that hybridize specifically to the anchors. Thepeptide/oligo linkers are self assembled on the surface by adding themto all wells of the MAPS plates.

[0258] For screening, the five kinases at appropriate concentrations (sothat the rates of phosphorylation of the substrates are balanced as muchas possible) are added to each well along with one of 8800 differentcompounds to be tested. The compounds are tested for their ability todirectly inhibit the isolated enzymes. The amount of phosphorylation ofeach arrayed peptide is detected by adding labeled antibodies that bindonly to peptides that are phosphorylated on tyrosine. Any wells thatshow a reduction in some of the phospho-tyrosine spots but not all ofthe spots are interesting. Compounds that had been added to those wellscan be tested further as possible selective inhibitors of some of thekinases tested.

[0259] The scheme of the assay is shown in the top panel of FIG. 17. Achimeric linker molecule is prepared in which a 25 base pairoligonucleotide complementary to one of the anchors is crosslinked to apeptide substrate of a tyrosine phosphokinase enzyme. The chimericoligo-peptide substrate self-assembles onto an array of oligonucleotideanchors, the kinase enzyme is used to phosphorylate the peptide portionof the chimera, and after the enzyme reaction is allowed to proceed, theamount of phosphorylation of the peptide is determined byanti-phoshotyrosine or anti-phosphoserine antibodies with an attacheddetection fluorophore or enzyme.

[0260] The results of the assay are shown in the lower panel. Thehomobifunctional crosslinker, DSS (Pierce), was used to attach the 5′amino group of an oligonucleotide linker to the N terminus of a peptidesynthesized with a phosphorylated tyrosine. The sequence of the peptidein single-letter code was: TSEPQpYQPGENL (SEQ ID: 32), where pYrepresents phosphotyrosine. The chimera was either used directly orfirst 10 brought to pH 14 for 60 minutes in order to partially hydrolyzethe phosphate group from the tyrosine. The phosphorylated or partiallydephosphorylated chimeric molecules were self-assembled ontocomplementary anchor molecules within a MAPS plate at the concentrationsshown for one hour. After washing and blocking the wells with 0.3% BSAin SSPTP antiphosphotyrosine antibody crosslinked to HRP (antibody 4G10from Upstate Biotechnology, Lake Placid, N.Y.) was added at a 1:3000dilution in SSPTP for one hour, and the amount of antibody attacheddetected with chemiluminescence substrate, Super Signal Blaze. The imageshown was accumulated on the CCD array for 1 minute. As expected adifference was seen in the amount of phosphate attached to theoligo-peptide. This difference is the basis for an assay measuring howactive a series of kinases is when treated with different possibleinhibitors.

Example 19 A Binding Assay for the Detection of Selective Inhibitors ofthe Interaction Between SH2 Domains and Phosphorylated Peptides

[0261] SH2 domains serve as docking subunits of some growth regulatoryproteins. The domains bind to phosphotyrosine containing proteins orpeptides with imperfect specificity. That is, some phosphotyrosinepeptides bind specifically to one or to few SH2 proteins while othersbind widely to many SH2 proteins.

[0262] For this assay, the linkers are phosphorylated peptidescovalently attached to oligonucleotides. The peptide moieties areselected for their ability to bind to a group of selected SH2 proteins.The linkers convert generic MAPS plates to plates with ligands specificfor the group of SH2 proteins. 100 96-well MAPS plates bearing theligands are generated. The proteins are isolated and labeled with, forexample, a cy5 fluorescent molecule.

[0263] In order to screen for inhibitors of the SH2domain/phosphopeptide interaction, the group of labeled SH2 proteins isadded to each well of the 100 96-well MAPS plates, and in each well adifferent test compound is added. Hence the effect of each compoundindividually on the interaction of the SH2 proteins with theirphosphopeptide ligands is tested. The assay is to measure thefluorescence of bound SH2 protein associated with each surface-boundpeptide linker. For any well showing reduced fluorescence at some spotsbut not all spots, the compound added can be further tested as aputative selective inhibitor of SH2 docking.

Example 20 High Throughput Screening (see FIG. 22)

[0264] Shown is a high throughput MAPS plate demonstrating the detectionof signal from 96 wells in a single experiment. Hybridization to thesame oligonucleotide was measured with 16 replicates in 80 wells. Asshown, the reproducibility of the 1280 hybridization assays was veryhigh. The left-most and right-most columns served as controls tostandardize the signal for different concentrations of theoligonucleotide.

[0265] In a similar fashion, 16 different oligonucleotides can be testedin each well, and the test repeated in the 80 different wells of theplate. Of course, an even greater number of different oligonucleotidesor other probes, (e.g., 100 nucleotide probes) can be assayed in eachwell, and many plates can be tested simultaneously (e.g., 100 plates,such as 96-well microtiter plates). The large number of assays which canbe performed on each sample (e.g., in the latter case, about 100different assays) and the large number of samples which can be assayedsimultaneously (e.g., in the latter case, about 96×100, or 9600different samples) provides for very high throughput.

Example 21 Preparation of Amplified Target (see FIG. 23)

[0266] A PCR primer (Primer One) is attached to a solid support (e.g., abead or a reaction vessel) via a chemical modification that has beenintroduced at the 5′ terminus of the primer oligonucleotide. The primercomprises, 5′ to 3′, the chemical modification, a restriction enzymesite, and a sequence that is complementary to a target of interest(e.g., a cDNA copy of an mRNA of interest). The target is amplified byPCR, using as PCR primers the attached Primer One plus a Primer Two,which comprises, 5′ to 3′, a sequence that is specific for a detectoroligonucleotide and a sequence that is complementary to a differentportion of the target than that of Primer One. Following PCRamplification, the amplified target DNA is washed to remove excessreaction material and is released from the solid support by cleavagewith a restriction enzyme specific for the restriction site on PrimerOne. The amplified primer is thus released into the liquid phase.Thermal and/or chemical procedures can be used to deactivate therestriction enzyme and to denature the double stranded DNA product. Thereleased, single stranded DNA target molecules can then be contactedwith a surface comprising anchors and/or linkers, and the target can bedetected using detector oligonucleotides complementary to thedetector-specific sequences of Primer Two.

Example 22 Preparation of Amplified Target

[0267] A PCR primer (Primer One) is attached to a solid support (e.g., abead or a reaction vessel) via a chemical modification that has beenintroduced into the 5′ terminus of the primer oligonucleotide. Theprimer comprises, 5′ to 3′, the chemical modification, a peptidesequence which can be cleaved by a protease, and a sequence which iscomplementary to a target of interest (e.g., a cDNA copy of an mRNA ofinterest). Instead of a peptide, any other element which can be cleavedspecifically can also be used. Following PCR amplification as described,e.g., in Example 21, the PCR product, still attached to the solidsupport, is denatured and (optionally) washed, leaving behind a singlestranded molecule attached to the support. The washed, attached,molecule can then be cleaved and released (e.g., by treatment with anappropriate protease), and contacted with a surface comprising anchorsand/or linkers. Alternatively, the strand of the amplified target whichis released following denaturation can be contacted with the surfacecomprising anchors and/or linkers. In either case, only one strand ofthe amplified target is contacted (e.g., hybridized) with a linker, socompetition for hybridization from the opposite strand of the amplifiedtarget is eliminated and background is reduced. Linkers can be designedto be specific for either, or both, of the amplified target strands.

Example 23 Assay with Detection Linkers and Reporter Agents (See FIG.24)

[0268] A sample comprising an mRNA of interest is subjected to anuclease protection procedure, using as a protection fragment anoligonucleotide which comprises a target specific moiety and a controloverhang moiety, which is not complementary to the mRNA. Followingnuclease digestion, the control overhang moiety can be cleaved off, asdesired, as is illustrated in the left portion of the figure; or theoverhang can fail to be digested, as is illustrated in the right portionof the figure. The resulting nuclease protection fragments arehybridized to a detection linker, which comprises a target-specificmoiety and a control overhang-specific moiety. In the assay shown in theleft part of the figure, the control overhang moiety of the detectionlinker remains unhybridized; by contrast, in the assay shown in theright part of the figure, the control overhang moiety of the detectionlinker hybridizes to the residual control overhang sequence of theprotection fragment. In a subsequent step of the assay, a reporterreagent, which comprises a moiety that can interact with controloverhang-specific moiety of the detection linker, is allowed to interactwith the complexes. In the assay shown in the left part of the figure,the reporter reagent hybridizes to the control overhang-specific moietyof the detection linker, which remains available for hybridization, andthe complex can be detected by virtue of the signaling entity on thereporter reagent. By contrast, in the assay shown in the right part ofthe figure, the reporter reagent is unable to bind to the complexbecause the complementary sequences are not available for hybridization,so no signal is associated with the complex.

[0269] In many of the assays of this invention, a reporter reagent caninteract with any sequence present in a detection linker, not limited toa sequence specific for a control overhang.

Example 24 Multiple Fluors (See FIG. 25)

[0270] A region comprising five loci, A-E, is shown in FIG. 25. Eachlocus comprises a different group of substantially identical anchors,anchors A-E. To the anchors at locus A are hybridized four differenttypes of linkers, each of which comprises a moiety specific for anchorA. However, each of the anchors comprises a different target-specificmoiety: for targets 1, 2, 3 or 4. Therefore, after hybridization oftargets to the anchor/linker complexes, targets 1, 2, 3, and 4 can allbecome localized at locus A. Similarly, four different types of linkerscan hybridize to locus B. Each linker comprises a moiety specific foranchor B, but the target-specific moieties are specific for targets 5,6, 7 or 8. In a similar fashion, targets 9-12 can become associated withlocus C, targets 13-16 at locus D, and targets 17-20 at locus E. If eachof these targets is labeled, either directly or indirectly, with adifferent, independently detectable fluor, such as, e.g., anupconvertable phosphore, one can independently detect all 20 targets atthe five indicated loci.

Example 25 An Assay in High Throughput Format

[0271] In this example, a transcription assay of the invention is usedto detect and quantify changes in a gene expression pattern, in a formatready for high throughput screening. All steps in the assay areperformed robotically. Routine washing steps are not explicitlydescribed. All reactions are carried out by conventional procedures,which are known in the art and/or described herein.

[0272] THP-1 human monocytes are grown in 96-well V-bottom microtiterplates, with 50,000 or 150,000 cells/well. The cells are eitheruntreated or are differentiated with phorbol 12-myristate 13-acetate(PMA) for 48 hours, followed by activation with lipopolysaccharide (LPS)for four hours. Following treatment, the cells are lysed in guanidiniumisothyocyanate and frozen until needed. mRNA is obtained usingstreptavidin-paramagnetic particles to which is bound biotin-poly dT.Alternatively, total RNA is obtained by extraction with tri-reagent(Sigma Chemical Co., St. Louis, Mo.). Samples comprising either mRNA ortotal RNA are subjected to a nuclease protection procedure, using as DNAprotection fragments a mixture of thirteen 60-mer single strandoligonucleotides, each of which comprises, 5′ to 3′, a 25-mer specificfor one of the thirteen targets of interest (GAPDH, IL-1, TNF-α,cathepsin G, cox-2, cyclin-2, vimentin, LD78-β, HMG-17, osteopontin,β-thromboglobin, angiotensin or actin); a 10-mer spacer; a 25-merspecific for a common oligonucleotide detector probe; and a 15-mercommon control overhang sequence. mRNA is thereby converted into astochiometric amount of “corresponding DNA protection fragment,” whichis detected in the assay. Control experiments in which thesecorresponding DNA protection fragments are incubated with a probespecific for the control overhang sequence show that substantially onlysequences specific for the mRNA targets of interest are present in thecorresponding protection fragments, as expected if nuclease digestionhas occurred as desired.

[0273] Surfaces are prepared according to the methods of the invention.In each well of a 96-well DNA Bind Plate is placed an array of sixteendifferent 25-mer oligonucleotide anchors. Fourteen different anchorspecies are used. One anchor species is used at three of the fourcorners of the array, and 13 different anchor species are used, one eachat the remaining locations in the array. The anchors are thenhybridized, in a defined orthogonal pattern, to 60-mer oligonucleotidelinkers, each of which comprises, 5′ to 3′, a 25-mer corresponding toone of the thirteen targets of interest, a 10-mer spacer, and a 25-merspecific for one of the anchors. Thus, in each of the multiply repeated16-spot arrays, each of the thirteen target-specific linkers islocalized at a defined position (locus) in the array. See FIG. 18 for anillustration of such an orthogonal array. Linkers corresponding toGAPDH, a constitutively expressed housekeeping gene which serves as aninternal normalization control, are represented at three loci withineach array. Control experiments indicate that the linkers, as well asthe protection fragments and detector oligonucleotides used in theexperiment, exhibit the desired specificity.

[0274] Samples comprising the mixtures of corresponding protectionfragments prepared as described above are hybridized to theanchor/linker arrays. Samples derived from either untreated or inducedcultures are used. The presence and amount of hybridized protectionfragments at each locus is then detected by hybridization to labeleddetector oligonucleotides. In order to normalize the amount of signal ateach locus, the detector oligonucleotides are diluted with appropriateamounts of blocked oligomers, as described herein. The amount of signalat each locus is processed and normalized to the control GAPDH signals.The data obtained are reproducible in eight replicate samples, as wellas in samples prepared from three independent experiments, performed ondifferent days. A summary of the relative abundance of the thirteentranscripts in one experiment is shown in the Table below. RelativeIntensity (10⁵ Cells/Well) Control Induced Gene Average CV (n = 16)Average CV (n = 16) Ratio GAPDH 10110  7% 9833  9% 0.97 IL-1 527 36%8124 38% 15.40 TNF 229 35% 2249 36% 9.80 GAPDH 9591 11% 10031 17% 1.05Cathepsin G 10394 31% 19648 46% 1.89 COX-2 415 39% 3557 25% 8.58Cyclin-2 1728 23% 2960 25% 1.71 Vimentin 25641 25% 71074 20% 2.77 LD781298 39% 13437 20% 10.35 HMG-17 8286 19% 2405 20% 0.29 Osteopontin 560442% 19053 46% 3.40 Thromboglobulin −53 — 31761 23% >100 GAPDH 10299 13%10136 12% 0.98 Angiotensin 3575 28% 6561 31% 1.84 Actin 12741 27% 2180223% 1.71 (blank) 108 — 234 —

Example 26 Computer Algorithm for Quantification of Multiple Array PlateData

[0275] A preferred algorithm finds the position of all spots for a MAPSplate and automatically calculates a best-fit estimate of the amplitudeof the signal for each data point.

[0276] Preferably, the algorithm is implemented by a computer program.

[0277] 1—Select a small part of the image data, a 40×40 box, containingthe intensity value of each pixel (picture element) of the image thatincludes the first well to be examined.

[0278] 2—Define a function that calculates the intensity expected ateach pixel position, using 16 unknowns. The unknowns are:

[0279] The amplitudes of each of 13 different microarray spots (that is,how bright are the real signals at each position of the DNA array).There are 13 of these for the 4×4 (=16) spots within each well becausesome of the 16 spots are duplicates of the same target.

[0280] The x offset and the y offset defining the exact position of the4×4 array of spots within this particular well

[0281] The background intensity of the picture within the well.

[0282] The function for each pixel position calculates the distancebetween the pixel and each spot, and adds up the contribution that eachspot makes to the intensity observed at that pixel, by multiplying thespot amplitude by the impulse response function for the given distance.For the images used the impulse response function is defined by the sumof a Gaussian and a Lorentzian of appropriate (constant) radii.

[0283] 3—Start the fitting for the current well by guessing the valuesof the parameters quickly. To do this, calculate the average imageintensity for 16 regions of the picture where the spots are expected tobe. Subtract an offset from these 16 averages, and scale the differenceby a constant factor. The offset and scaling constant are definedempirically. Rearrange the results to match up the 16 spots with the 13amplitudes. For the background and offets use any small numbers.

[0284] 4—Optimize the fitted values (for the 16 unknowns) by curvefitting. In particular use a non-linear least squares algorithm withMarquadt procedure for linearizing the fitting function, fitting 16unknowns to 40×40=1600 equations (although of course not all equationsare linearly independent).

[0285] 5—Use the x,y offset as fitted for the current well to estimatewith improved precision where the grid will be for the next well of themicroplate. It is expected to be 9 millimeters offset relative to thenext neighbor well (converted to distance in the number of pixels by themagnification factor of the imaging system). Since the distance betweenwells is small relative to the size of the plate, using local estimatesof position is most accurate.

[0286] 6—With the improved estimate of position, define a smaller box ofimage for the next well, moving to a 30×30 box of pixels. This makes thefitting proceed more quickly.

[0287] Go back to step 2 and repeat for each well.

Example 27 High Throughput Screening (See FIGS. 26 and 27)

[0288]FIG. 26 illustrates raw image data for an assay using detectionlinkers and a single reporter reagent. The assay tests the expression of13 native mRNA species from 96 different cell samples. Each well of a96-well plate contains 10⁵ THP-1 cells untreated (left half of thefigure), or induced to monocytes with PMA and LPS (right half).

[0289] The pattern of expression changes consistently. IL-1, TNF, COX-2,Vimentin, LD78, Osteopontin, and beta-Thromboglobulin are induced.Cathepsin-G, Cyclin-b, HMG-17 and Angiotensin are turned off. GAPDH andActin are unchanged. FIG. 27 presents the spatial arrangement of genesfor the THP-1 cells, along with two sample wells of data (selected fromFIG. 26).

[0290] The oligos used in this experiments are listed below. For sometargets, the intensity of signal is reduced by diluting the detectionlinker with an incomplete detection linker oligonucleotide, containingthe 25 bases complementary to the protection fragment but not containingthe sequence complementary to the reporter reagent. These incompleteoligos are referred to as “attenuation factors.”

[0291] Table 1 presents quantification of the raw data presented above.This screening assay is done in high throughput fashion. Cells are grownand treated in 96-well plates at an average of 10⁵ cells/well, thenumber of cells that can be conveniently handled in microplates. Anexpression pattern for 13 genes is measured in high throughput format,from small cell samples. The results obtained using assay corroborateand extend the literature, as summarized in Table 1. The assay candetect less than one copy per cell. The literature references reflectobservations accumulated from a variety of related cell types, such asU-937 cells. The large differences seen between the Control and Induced20 conditions result from the very low background signals for ourmeasurements. The use of detection linkers, allowing for only onespecies of reporter reagent helps to reduce the background for theassay. This is because the total concentration of HRP—containingreporter reagent is much reduced. TABLE 1 Relative Abundance (RNAMolecules per cell⁺) MAPS 96-16 Format, 10⁵ Cells/Well Gene ControlInduced Literature GAPDH  30 ± *7% 30 ± 14% No Change IL1-beta **nd 684± 14%  Increase TNF 3.0 ± 40% 214 ± 23%  Increase Cathepsin-G  53 ± 8% nd Decrease COX-2 nd 8.3 ± 23%  Increase Cyclin-2 2.8 ± 11% 0.5 ± 46% No Change Vimentin nd 37 ± 33% Increase LD78-b nd 3360 ± 28%  IncreaseHMG-17  336 ± 5%  33 ± 23% Decrease Osteopontin nd 18 ± 23% Not ReportedThromboglobulin nd 66 ± 15% Increase Angiotensin 0.5 ± 18% 0.1 ± 66% Not Reported Actin  79 ± 7%  43 ± 21% No Change

[0292] The oligos used in Example 27 with detection linkers and a singleprobe are: Fixed Probe: CACCTCCAAACAGTGAAGGAGAGCA (SEQ ID:33)(conjugated to HRP) Target #1; ID:MI7851GAPDH (572-513) Anchor: -length= 25 CGCCGGTCGAGCGTTGTGGGAGCGC (SEQ ID:34) Target: -length = 60TGAGAAGTATGACAACAGCCTCAAGATCATCAGCAAT (SEQ ID:35)GCCTCCTGCACCACCAACTGCTT Linker: -length = 60ATGCCTCCTGCACCACCAACTGCTTGATACTGAGTGC (SEQ ID:36)GCTCCCACAACGCTCGACCGGCG Protection Fragment: -length = 75AAGCAGTTGGTGGTGCAGGAGGCATTGCTGATGATCT (SEQ ID:37)TGAGGCTGTTGTCATACTTCTCAGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTTG (SEQ ID:38)AGAAGTATGACAACAGCCTCAAG Attenuation Factor: -length = 25TGAGAAGTATGACAACAGCCTCAAG (SEQ ID:39) Target #2; ID:M 15840 ILI-beta(4392-4333) Anchor: -length = 25 TCCACGTGAGGACCGGACGGCGTCC (SEQ ID:40)Target: -length = 60 CGACACATGGGATAACGAGGCTTATGTGCACGATGCA (SEQ ID:41)CCTGTACGATCACTGAACTGCAC Linker: -length = 60CACCTGTACGATCACTGAACTGCACGATACTGAGTGG (SEQ ID:42)ACGCCGTCCGGTCCTCACGTGGA Protection Fragment: -length = 75GTGCAGTTCAGTGATCGTACAGGTGCATCGTGCACAT (SEQ ID:43)AAGCCTCGTTATCCCATGTGTCGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCG (SEQ ID:44)ACACATGGGATAACGAGGCTTAT Attenuation Factor: -length = 25CGACACATGGGATAACGAGGCTTAT (SEQ ID:45) Target #3; ID:M1O988TNF (780-721)Anchor: -length = 25 CACTACGGCTGAGCACGTGCGCTGC (SEQ ID:46) Target:-length = 60 CGGAACCCAAGCTTAGAACTTTAAGCAACAAGACCAC (SEQ ID:47)CACTTCGAAACCTGGGATTCAGG Linker: -length = 60ACCACTTCGAAACCTGGGATTCAGGGATACTGAGTGC (SEQ ID:48)AGCGCACGTGCTCAGCCGTAGTG Protection Fragment: -length = 75CCTGAATCCCAGGTTTCGAAGTGGTGGTCTTGTTGCT (SEQ ID:49)TAAAGTTCTAAGCTTGGGTTCCGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCG (SEQ ID:50)GAACCCAAGCTTAGAACTTTAAG Attenuation Factor: -length = 25CGGAACCCAAGCTTAGAACTTTAAG (SEQ ID:51) Target #4; ID:M17851GAPDH(572-513) (same as for Target #1) Target #5; ID:M16117Cathepsin-G(373-314) Anchor: -length = 25 GAACCGCTCGCGTGTTCTACAGCCA (SEQ ID:52)Target: -length = 60 GCGGACCATCCAGAATGACATCATGTTATTGCAGCTG (SEQ ID:53)AGCAGAAGAGTCAGACGGAATCG Linker: -length = 60TGAGCAGAAGAGTCAGACGGAATCGGATACTGAGTTG (SEQ ID:54)GCTGTAGAACACGCGAGCGGTTC Protection Fragment: -length = 75CGATTCCGTCTGACTCTTCTGCTCAGCTGCAATAACA (SEQ ID:55)TGATGTCATTCTGGATGGTCCGCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGC (SEQ ID:56)GGACCATCCAGAATGACATCATG Attenuation Factor length = 25GCGGACCATCCAGAATGACATCATG (SEQ ID:57) Target #6; ID:M90100COX-2(240-181) Anchor: -length = 25 CTCGTTCCGCGTCCGTGGCTGCCAG (SEQ ID:58)Target: -length = 60 CCGAGGTGTATGTATGAGTGTGGGATTTGACCAGTAT (SEQ ID:59)AAGTGCGATTGTACCCGGACAGG Linker: -length = 60ATAAGTGCGATTGTACCCGGACAGGGATACTGAGTCT (SEQ ID:60)GGCAGCCACGGACGCGGAACGAG Protection Fragment: -length = 75CCTGTCCGGGTACAATCGCACTTATACTGGTCAAATC (SEQ ID:61)CCACACTCATACATACACCTCGGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCC (SEQ ID:62)GAGGTGTATGTATGAGTGTGGGA Attenuation Factor: -length = 25CCGAGGTGTATGTATGAGTGTGGGA (SEQ ID:63) Target #7; ID:M74091 cyclin(932-873) Anchor: -length = 25 CGGTCGGCATGGTACCACAGTCCGC (SEQ ID:64)Target: -length = 60 CACCTCCAAACAGTGAAGGAGAGCAGGGTCCAAATGG (SEQ ID:65)AAGTCAGAACTCTAGCTACAGCC Linker: -length = 60GGAAGTCAGAACTCTAGCTACAGCCGATACTGAGTGC (SEQ ID:66)GGACTGTGGTACCATGCCGACCG Protection Fragment: -length 75GGCTGTAGCTAGAGTTCTGACTTCCATTTGGACCCTG (SEQ ID:67)CTCTCCTTCACTGTTTGGAGGTGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:68)CCTCCAAACAGTGAAGGAGAGCA Attenuation Factor: -length = 25CACCTCCAAACAGTGAAGGAGAGCA (SEQ ID:69) Target #8; ID:M14144vimentin(1338-1279) Anchor: -length = 25 GCGCGCCGCGTTATGCATCTCTTCG (SEQ ID:70)Target: -length = 60 GTGGATGCCCTTAAAGGAACCAATGAGTCCCTGGAAC (SEQ ID:71)GCCAGATGCGTGAAATGGAAGAG Linker: -length = 60ACGCCAGATGCGTGAAATGGAAGAGGATACTGAGTCG (SEQ ID:72)AAGAGATGCATAACGCGGCGCGC Protection Fragment: -length = 75CTCTTCCATTTCACGCATCTGGCGTTCCAGGGACTCA (SEQ ID:73)TTGGTTCCTTTAAGGGCATCCACGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGT (SEQ ID:74)GGATGCCCTTAAAGGAACCAATG Attenuation Factor: -length = 25GTGGATGCCCTTAAAGGAACCAATG (SEQ ID:75) Target #9; ID:D90145 LD78-b(2049-1990) Anchor: -length = 25 GTTAGCATACGTGTCACCACACCGG (SEQ ID:76)Target: -length = 60 CACCTCCCGACAGATTCCACAGAATTTCATAGCTGAC (SEQ ID:77)TACTTTGAGACGAGCAGCCAGTG Linker: -length = 60ACTACTTTGAGACGAGCAGCCAGTGGATACTGAGTCC (SEQ ID:78)GGTGTGGTGACACGTATGCTAAC Protection Fragment: -length = 75CACTGGCTGCTCGTCTCAAAGTAGTCAGCTATGAAAT (SEQ ID:79)TCTGTGGAATCTGTCGGGAGGTGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:80)CCTCCCGACAGATTCCACAGAAT Attenuation Factor: -length = 25CACCTCCCGACAGATTCCACAGAAT (SEQ ID:81) Target #10; ID:X13546 HMG-17M12623-mRNA (191-132) Anchor: -length = 25 CGTCAGTCCGTCGGCCAGCTCTTCC(SEQ ID:82) Target: -length = 60 CAAAGGTGAAGGACGAACCACAGAGAAGATCCGCGAG(SEQ ID:83) GTTGTCTGCTAAACCTGCTCCTC Linker: -length = 60AGGTTGTCTGCTAAACCTGCTCCTCGATACTGAGTGG (SEQ ID:84)AAGAGCTGGCCGACGGACTGACG Protection Fragment: -length = 75GAGGAGCAGGTTTAGCAGACAACCTCGCGGATCTTCT (SEQ ID:85)CTGTGGTTCGTCCTTCACCTTTGGCTTGTCTAAGTCT G Detection-Linker: -length 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:86)AAGGTGAAGGACGAACCACAGAG Attenuation Factor: -length = 25CAAAGGTGAAGGACGAACCACAGAG (SEQ ID:87) Target #11; ID:X13694 Osteopontin(783-724) Anchor: -length = 25 ATCCAGTTAACCACATGCTAGTACC (SEQ ID:88)Target: -length = 60 CCGTGGGAAGGACAGTTATGAAACGAGTCAGCTGGAT (SEQ ID:89)GACCAGAGTGCTGAAACCCACAG Linker: -length = 60ATGACCAGAGTGCTGAAACCCACAGGATACTGAGTGG (SEQ ID:90)TACTAGCATGTGGTTAACTGGAT Protection Fragment: -length = 75CTGTGGGTTTCAGCACTCTGGTCATCCAGCTGACTCG (SEQ ID:91)TTTCATAACTGTCCTTCCCACGGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCC (SEQ ID:92)GTGGGAAGGACAGTTATGAAACG Attenuation Factor: -length = 25CCGTGGGAAGGACAGTTATGAAACG (SEQ ID:93) Target #12;ID:M17017b-thromboglobulin (142-83) Anchor: -length = 25TTAGCGTTGGCCGAGGTTCATAGCC (SEQ ID:94) Target. -length = 60GTGTAAACATGACTTCCAAGCTGGCCGTGGCTCTCTT (SEQ ID:95)GGCAGCCTTCCTGATTTCTGCAG Linker: -length = 60TTGGCAGCCTTCCTGATTTCTGCAGGATACTGAGTGG (SEQ ID:96)CTATGAACCTCGGCCAACGCTAA Protection Fragment: -length = 75CTGCAGAAATCAGGAAGGCTGCCAAGAGAGCCACGGC (SEQ ID:97)CAGCTTGGAAGTCATGTTTACACGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGT (SEQ ID:98)GTAAACATGACTTCCAAGCTGGC Attenuation Factor: -length = 25GTGTAAACATGACTTCCAAGCTGGC (SEQ ID:99) Target #13; ID:M17851GAPDH(572-513) (same as for Target #1) Target #14; ID:K02215 angiotensin(805-746) Anchor: -length = 25 CATTACGAGTGCATTCGCATCAAGG (SEQ ID:100)Target: -length = 60 CACGCTCTCTGGACTTCACAGAACTGGATGTTGCTGC (SEQ ID:101)TGAGAAGATTGACAGGTTCATGC Linker: -length = 60GCTGAGAAGATTGACAGGTTCATGCGATACTGAGTCC (SEQ ID:102)TTGATGCGAATGCACTCGTAATG Protection Fragment: -length = 75GCATGAACCTGTCAATCTTCTCAGCAGCAACATCCAG (SEQ ID:103)TTCTGTGAAGTCCAGAGAGCGTGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:104)CGCTCTCTGGACTTCACAGAACT Attenuation Factor: -length = 25CACGCTCTCTGGACTTCACAGAACT (SEQ ID:105) Target #15; ID:MI0277Actin(2627-2568) Anchor: -length = 25 ATCATGTAAGTCTTCGGTCGGTGGC (SEQ ID:106)Target: -length = 60 GAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCA (SEQ ID:107)TCATGAAGTGTGACGTGGACATC. Linker: -length = 60CATCATGAAGTGTGACGTGGACATCGATACTGAGTGC (SEQ ID:108)CACCGACCGAAGACTTACATGAT Protection Fragment: -length = 75GATGTCCACGTCACACTTCATGATGGAOTTGAAGGTA (SEQ ID:109)GTTTCGTGGATGCCACAGGACTCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGA (SEQ ID:110)GTCCTGTGGCATCCACGAAACTA Attenuation Factor: -length = 25GAGTCCTGTGGCATCCACGAAACTA (SEQ ID:111) Target #16; (this spot is notused)

Example 28 Simultaneous Detection of DNA and RNA (See FIG. 29)

[0293] THP-1 human monocytes are grown in 96-well V-bottom microtiterplates, with 30,000 to 150,000 cells/well. Control cells which have notbeen differentiated with PMA 30 or activated with LPS are used, becausethe RNAs for certain genes (e.g., IL-2, Cox-2, LD78, Osteopontin andThromboglobulin) are not present in those cells and therefore only DNAis measured in the assay; whereas both DNA and RNA for GAPDH are presentand measured. The cells are heated to 105° C. in an aqueous medium(lysis buffer), and nuclease protection fragments specific for thetargets of interest are added to the lysates. Lysis at elevatedtemperature releases DNA in a measurable form, as well as RNA. For themeasurement of DNA, the nuclease protection fragments added are thosefor IL-1, Cox-2, LD78, Osteopontin and Thromboglobulin. For themeasurement of both DNA and RNA, the preceding nuclease protectionfragments, as well as one specific for GAPDH, are added. Nucleaseprotection reactions are performed in the wells as described elsewhereherein.

[0294] The arrays are formed and the hybridization of protectionfragments is performed essentially as described in Example 27. Detectionof DNA vs DNA+RNA is done by serial hybridization of detection linkers.Serial hybridization is performed here in order to balance the signalsfrom RNA and DNA targets (as discussed below); serial hybridization is,of course, not a requirement for assays in which DNA and RNA targets aredetected together. In the first round, detection linkers for IL-2,Cox-2, LD78, Osteopontin and Thromboglobulin are added. In the secondround, the detection linker for GAPDH is added. Serial hybridization isperformed in order to image the DNA signal, which is relatively muchweaker than the RNA signal due to much lower copy number per sample, fora longer period of time in order to accumulate a higher signalintensity.

[0295] The results are presented in FIG. 29. The left panel illustratesthat when genomic DNA alone is examined, the genomic sequencestested—IL-1, Cox-2, LD78, Osteopontin and Thromboglobulin—can all bedetected at the appropriate loci, and are present in approximately thesame amounts. That genomic DNA is being measured is indicated by Table1, which shows that in such control cells, RNA for these genes is notdetectable. The right panel, in which both the DNA and RNA are measured,but a much shorter image exposure time is collected such that the DNAsignal is much weaker than in the left panel, shows that when DNA andRNA are examined together, the control genomic sequences can be detectedas before, as internal normalization standards, and the expressedgene—GAPDH—is present at a much higher level than the controls. Byquantitating the relative amounts of signal in the controls and theexpressed GAPDH mRNA, one can calculate the amount of mRNA expressed percell.

Example 29 Detection of Expressed SNPs (See FIGS. 30 and 31)

[0296]FIG. 30 schematically illustrates one type of assay for expressedSNPs. Here, a nuclease protection fragment is designed to hybridize tothe region of an RNA containing a SNP, in such a manner that when anappropriate enzyme (e.g., RNAse H) is added, if the nuclease protectionfragment has hybridized to RNA for which there is a mis-matched base(here, the SNP), the enzyme will cleave the nuclease protectionfragment. In this example, the resulting cleaved fragment cannothybridize to the array (e.g., due to the hybridization conditions suchas temperature used). In other embodiments, hybridization to the arraycan occur, but a detection linker cannot bind to the cleaved protectionfragment (e.g., due to the hybridization conditions such as temperatureused); or the cleavage occurs in such a way that the cleaved protectionfragment cannot hybridize simultaneously to the array and to a detectionlinker.

[0297]FIG. 31 illustrates the results of such an assay, performed byconventional methods as described herein. Wild type actin is used as aninternal control, and a protection fragment corresponding to GAPDHcontaining an engineered SNP is differentiated from a protectionfragment corresponding to wild type GAPDH. FIG. 31 depicts themeasurement of multiple samples containing either wild type GAPDH andwild type actin (left column and left panel of blow-up) or containingSNP GAPDH and wild type actin (right column and right panel of blow-up).The center panel of the blow-up depicts the array layout.

Example 30 High Throughput Screening (See FIGS. 32-35)

[0298] Transcription assays are performed essentially as described inExample 28, except, e.g., the anchors are placed on irradiated platesrather than DNA Bind plates. The same anchors described in Example 27are used, but certain targets in the array are changed, namely, only oneanchor is used to measure GAPDH, and Tubulin, actin, and LDH are added.The other targets measured are IL-1, TNF-a, Cathepsin G, Cox-2, G-CSF,GM-CSF, GST-Pi1, HMG-17, Cyclophilin, b-Thromboglobulin, TIMP-1, MMP-9.The array is depicted in FIG. 32 and the linker and nuclease protectionfragment sequences are given below. Approximately 30,000 cells are usedper well (not adjusted for continued proliferation of control cellsduring the course of PMA and LDH treatment of the treated cells).

[0299] Sequences: Target #1 GAPDH (same as Target #1 example 27) Target#2 IL-lb (same as Target #2 Example 27) Target #3 TNF-a (same as Target#3 Example 27) Target #4 Tubulin (AF141347) Anchor: -length = 25TAAGCGTCTCTAGGAAGGGACGTGG (SEQ ID:112) Target: -length = 60GACGTGGTTCCCAAAGATGTCAATGCTGCCATTGCCA (SEQ ID:113)CCATCAAGACCAAGCGTACCATC Linker: -length = 60CACCATCAAGACCAAGCGTACCATCGATACTGAGTCC (SEQ ID:114)ACGTCCCTTCCTAGAGACGCTTA Protection Fragment: -length = 75GATGGTACGCTTGGTCTTGATGGTGGCAATGGCAGCA (SEQ ID:115)TTGACATCTTTGGGAACCACGTCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGA (SEQ ID:116)CGTGGTTCCCAAAGATGTCAATG Attenuation Factor: -length = 25GACGTGGTTCCCAAAGATGTCAATG (SEQ ID:117) Target #5 Cathepsin-G (same asTarget #5 Example 27) Target #6 Cox-2 (same as Target #6 Example 27)Target #7 G-CSF (E01219) Anchor: -length = 25 CGGTCGGCATGGTACCACAGTCCGC(SEQ ID:118) Target: -length = 60 GAGGGAGCAGACAGGAGGAATCATGTCAGGCCTGTGT(SEQ ID:119) GTGAAAGGAAGCTCCACTGTCAC Linker: -length = 60GTGTGAAAGGAAGCTCCACTGTCACGATACTGAGTGC (SEQ ID:120)GGACTGTGGTACCATGCCGACCG Protection Fragment: -length = 75GTGACAGTGGAGCTTCCTTTCACACACAGGCCTGACA (SEQ ID:121)TGATTCCTCCTGTCTGCTCCCTCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGA (SEQ ID:122)GGGAGCAGACAGGAGGAATCATG Attenuation Factor: -length = 25GAGGGAGCAGACAGGAGGAATCATG (SEQ ID:123) Target #8 GM-CSF (E02975) Anchor:-length = 25 GCGCGCCGCGTTATGCATCTCTTCG (SEQ ID:124) Target: -length = 60CACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTT (SEQ ID:125)CCTGTGCAACCCAGATTATCACC Linker: -length = 60TTCCTGTGCAACCCAGATTATCACCGATACTGAGTCG (SEQ ID:126)AAGAGATGCATAACGCGGCGCGC Protection Fragment: -length = 75GGTGATAATCTGGGTTGCACAGGAAGTTTCCGGGGTT (SEQ ID:127)GGAGGGCAGTGCTGCTTGTAGTGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:128)CTACAAGCAGCACTGCCCTCCAA Attenuation Factor: -length = 25CACTACAAGCAGCACTGCCCTCCAA (SEQ ID:129) Target #9 GST-PI1 X06547 Anchor:-length = 25 GTTAGCATACGTGTCACCACACCGG (SEQ ID:130) Target: -length = 60CAGGGAGGCAAGACCTTCATTGTGGGAGACCAGATCT (SEQ ID:131)CCTTCGCTGACTACAACCTGCTG Linker: -length = 60CTCCTTCGCTGACTACAACCTGCTGGATACTGAGTCC (SEQ ID:132)GGTGTGGTGACACGTATGCTAAC Protection Fragment: -length = 75CAGCAGGTTGTAGTCAGCGAAGGAGATCTGGTCTCCC (SEQ ID:133)ACAATGAAGGTCTTGCCTCCCTGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:134)GGGAGGCAAGACCTTCATTGTGG Attenuation Factor: -length = 25CAGGGAGGCAAGACCTTCATTGTGG (SEQ ID:135) Target #10 HMG-17 (same as Target#10 Example 27) Target #11 Cyclophilin X52851 Anchor: -length 25ATCCAGTTAACCACATGCTAGTACC (SEQ ID:136) Target: -length 60GGGTTTATGTGTCAGGGTGGTGACTTCACACGCCATA (SEQ ID:137)ATGGCACTGGTGGCAAGTCCATC Linker: -length 60TAATGGCACTGGTGGCAAGTCCATCGATACTGAGTGG (SEQ ID:138)TACTAGCATGTGGTTAACTGGAT Protection Fragment: -length = 75GATGGACTTGCCACCAGTGCCATTATGGCGTGTGAAG (SEQ ID:139)TCACCACCCTGACACATAAACCCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGG (SEQ ID:140)GTTTATGTGTCAGGGTGGTGACT Attenuation Factor: -length = 25GGGTTTATGTGTCAGGGTGGTGACT (SEQ ID:141) Target #12 b-Thromboglobulin(same as Target #12 Example 27) Target #13 LDH X02152 Anchor: -length= 25 TCTCGGTCTGGAACGCCCGGCAACT (SEQ ID:142) Target: -length = 60GGTGGTTGAGAGTGCTTATGAGGTGATCAAACTCAAA (SEQ ID:143)GGCTACACATCCTGGGCTATTGG Linker: -length = 60AAGGCTACACATCCTGGGCTATTGGGATACTGAGTAG (SEQ ID:144)TTGCCGGGCGTTCCAGACCGAGA Protection Fragment: -length = 75CCAATAGCCCAGGATGTGTAGCCTTTGAGTTTGATCA (SEQ ID:145)CCTCATAAGCACTCTCAACCACCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGG (SEQ ID:146)TGGTTGAGAGTGCTTATGAGGTG Attenuation Factor: -length = 25GGTGGTTGAGAGTGCTTATGAGGTG (SEQ ID:147) Target #14 TIMP-1 X03124 Anchor:-length = 25 CATTACGAGTGCATTCGCATCAAGG (SEQ ID:148) Target: -length = 60CACCAAGACCTACACTGTTGGCTGTGAGGAATGCACA (SEQ ID:149)GTGTTTCCCTGTTTATCCATCCC Linker: -length = 60CAGTGTTTCCCTGTTTATCCATCCCGATACTGAGTCC (SEQ ID:150)TTGATGCGAATGCACTCGTAATG Protection Fragment: -length = 75GGGATGGATAAACAGGGAAACACTGTGCATTCCTCAC (SEQ ID:151)AGCCAACAGTGTAGGTCTTGGTGGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTCA (SEQ ID:152)CCAAGACCTACACTGTTGGCTGT Attenuation Factor: -length = 25CACCAAGACCTACACTGTTGGCTGT (SEQ ID:153) Target #15 MMP-9 J05070 Anchor:-length = 25 ATCATGTAAGTCTTCGGTCGGTGGC (SEQ ID:154) Target: -length 60GCAACGTGAACATCTTCGACGCCATCGCGGAGATTGG (SEQ ID:155)GAACCAGCTGTATTTGTTCAAGG Linker: -length = 60GGGAACCAGCTGTATTTGTTCAAGGGATACTGAGTGC (SEQ ID:156)CACCGACCGAAGACTTACATGAT Protection Fragment: -length = 75CCTTGAACAAATACAGCTGGTTCCCAATCTCCGCGAT (SEQ ID:157)GGCGTCGAAGATGTTCACGTTGCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGC (SEQ ID:158)AACGTGAACATCTTCGACGCCAT Attenuation Factor:-length = 25GCAACGTGAACATCTTCGACGCCAT (SEQ ID:159) Target #16 Actin M10277 Anchor:-length = 25 CTGAGTCCTCCGGTGCCTACGTGGC (SEQ ID:160) Target: -length = 60GAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCA (SEQ ID:161)TCATGAAGTGTGACGTGGACATC Linker: -length = 60CATCATGAAGTGTGACGTGGACATCGATACTGAGTGC (SEQ ID:162)CACGTAGGCACCGGAGGACTCAG Protection Fragment: -length = 75GATGTCCACGTCACACTTCATGATGGAGTTGAAGGTA (SEQ ID:163)GTTTCGTGGATGCCACAGGACTCGCTTGTCTAAGTCT G Detection-Linker: -length = 60TGCTCTCCTTCACTGTTTGGAGGTGGATACTGAGTGA (SEQ ID:164)GTCCTGTGGCATCCACGAAACTA Attenuation Factor: -length = 25GAGTCCTGTGGCATCCACGAAACTA (SEQ ID:165)

[0300]FIG. 33 shows that the reproducibility of the assay is high,providing a %CV range of about 3% to 13% when 30,000 cells are analyzed.

[0301]FIG. 34 shows that the sensitivity of the assay is high, e.g., thetarget mRNAs for GAPDH can be detected when RNA from a sample of as fewas, or fewer than, 1,000 cells is assayed.

[0302]FIG. 35, performed using essentially the protocol of Example 25and the array and targets of Example 27, shows that many of the targetmRNAs tested can be detected even when RNA from a sample of as few as,or fewer than, 1000 cells is assayed, and all of the targets, even thoseexpressed at low abundance, can be detected when the RNA is from as fewas, or fewer than, 10,000 cells.

Example 31 Oligonucleotide Reagent Options (See FIG. 36)

[0303]FIG. 36 schematically shows several types of oligonucleotidereagents which can be used in the methods of the invention. The figuredepicts assay schemes in which oligonucleotide anchors are attached to asurface via either their 5′ or their 3′ (“Inverted”) termini, and inwhich oligonucleotides have two recognition moieties that are eitheradjacent to each other (“Shortened”) or are separated by nucleic acidspacers. Each box represents 5 nucleotides.

[0304] Example 32

Nuclease Protection Fragment Amplification Methods (See FIGS. 37-39 and42)

[0305]FIG. 37 illustrates nuclease protection fragment amplification byPCR.

[0306]FIG. 38 illustrates nuclease protection fragment amplification byligase. By selecting a′ as a 12 base sequence, out of the 25 bases whichin ligated a′ a binds to the array, hybridization conditions can beselected which only allow the ligated, 25 base sequence of the a′amolecules to bind. Discrimination can be improved by using a modifiednucleotide(s) in each portion of the linker binding region of a′ and a.If the cycles of heat dissociation destroy the ligase, it can bere-added for each cycle.

[0307]FIG. 39 illustrates nuclease protection fragment amplification bynuclease protection. (DNA a) strand complementary to the (NucleaseProtection Fragment b) can contain modified bases which hybridize atlower temperature than the (RNA a), or the (RNA a) can be destroyedbefore (DNA a) is added. Likewise, the (Linker a) for (DNA a) cancontain modified nucleotides which allow hybridization at lowertemperature than (Nuclease Protection Fragment b), even if the (Linkera)/(DNA a) hybrid is 25 bases and the (DNA a)/(Nuclease ProtectionFragment b) is a 50 base hybridization, especially when taken intoaccount experimentally is that the DNA strands in solution are dilute,and passing thorough a highly concentrated, essentially infinitely highconcentration of (Linker A). The flow through apparatus can be replacedwith a plate comprising an array for capture. The linear array can bereplaced with a 2-D or 3-D array.

[0308]FIG. 42 illustrates nuclease protection fragment amplification bypolymerase. After the nuclease protection reaction is complete and thenuclease protection fragment is dissociated from the RNA, a primer (a′)is added which contains a double stranded promoter for an RNA polymerase(e.g., T7 polymerase) and a primer for extension along the nucleaseprotection fragment template (e.g., extension by reverse transcriptase(RT) for the replication of the RNA or DNA, or Taq polymerase for thereplication of DNA), such that after binding to the nuclease protectionfragment (e.g., RT or Taq polymerase) will use this as a template toform a double stranded DNA complex, with the double stranded promoterregion apposed to the end of the nuclease protection fragment sequence.Addition of ligase will ligate the second strand of the promoter to thenuclease protection fragment strand, unless the first base was amismatched SNP, and therefore during the nuclease protection reaction S1clipped off the unprotected base. Ligase will not ligate the promoter tothe nuclease protection fragment because of the skipped base. In thecase of ligation, the b strand is converted to an extended b″ strandincorporating the polymerase promoter and amplification can proceedusing polymerase, and continue as after the addition of RT primer b′.The promoter/extended end of the nuclease protection fragment is used tobind to the array or to a detection linker or detection probe. Fordetection of SNP, this hybridization is arranged so that the SNP site isapproximately in the middle of the sequence used for hybridization tothe array (detection linker, or detection probe, etc.). The array(detection linker, or detection probe) hybridization region of theextended nuclease protection probe does not have to include all thebases at the end (some of the bases at the end of the extended nucleaseprotection probe can overhang without having a complementaryhybridization sequence in the linker, etc.). In variations not depicted,it is not necessary to ligate before use of (e.g., T7) polymerase, andtherefore the SNP detection is performed by selecting a sequencepositioning the SNP in the middle of the sequence hybridizing to a′, andusing an SNP detection protocol as described elsewhere herein. It is notnecessary to extend the a′ strand, but instead RNA polymerase can usethe promoter hybridized to the single stranded nuclease protectionfragment (omitting the indicated RT extension, or alternative Taqpolymerase extension step depicted) to produce RNA.

[0309] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the invention to adapt it to various usage andconditions.

[0310] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The preceding preferred specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.

[0311] The entire disclosure of all applications, patents andpublications, cited above and in the figures are hereby incorporated byreference.

What is claimed is:
 1. A method of detecting at least one nucleic acidtarget, comprising contacting a sample which may comprise said target(s)with a nuclease protection fragment(s) specific for and which binds tosaid target(s), exposing the sample to a nuclease effective to digestsingle stranded nucleic acid, and contacting the resultant sample with acombination which comprises, before the addition of said sample, i) asurface comprising multiple spatially discrete regions, at least two ofwhich are substantially identical, each region comprising ii) at leasttwo different loci of oligonucleotide anchors, each anchor inassociation with iii) a bifunctional linker which has a first portionthat is specific for the oligonucleotide anchor, and a second portionthat comprises a probe which is specific for said nuclease protectionfragment(s), under conditions effective for said nuclease protectionfragment(s) to bind to said combination, wherein at least one locus inat least one of said regions is a “mixed locus,” which comprises about 2to about 4 different anchors, each having a specificity for a differentbifunctional linker, and wherein two or more of said different anchorslocated in said mixed locus are each in association with about 2 toabout 4 different bifunctional linkers, having different targetspecificities, hybridizing said bound protection fragments with specificdetection linkers, at least one of which is a “blocked” detection linkerand wherein at least one of said detection linkers is specific for afirst protection fragment which is associated with a first of said about2 to about 4 different bifunctional linkers, detecting said detectionlinkers with a specific reporter reagent which comprises an enzyme thatgenerates a chemiluminescent signal, stopping said signal by adjustingthe pH of the reaction mixture, hybridizing said bound protectionfragments with specific detection linkers, at least one of which is a“blocked” detection linker and wherein at least one of said detectionlinkers is specific for a second protection fragment which is associatedwith a second of said about 2 to about 4 different bifunctional linkers,and detecting said detection linkers with a specific reporter reagentwhich comprises an enzyme that generates a chemiluminescent signal.